- Email: [email protected]
Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis
Biochemical and Biophysical Research Communications xxx (2018) 1e7
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis Guangwei Zeng, Hui Liu, Haiyan Wang* Department of Cardiology, Tangdu Hospital, The Fourth Millitary University, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2018 Accepted 16 May 2018 Available online xxx
Apoptosis and mitochondria dysfunction are key contributors to myocardial ischemia-reperfusion (MI-R) injury. SIRT4, a mitochondrial-localized sirtuin, controls cellular energy metabolism and stress response, and is abundantly present in the heart, however, its role in MI-R injury is not clear. In the current study, we demonstrate that SIRT4 is downregulated in cardiomyocytes both in vitro and in vivo models after MI-R. Functionally, SIRT4 overexpression decreases myocardial infarct size and serum creatine phosphokinase (CPK) level, and vice versa, SIRT4 depletion by siRNA increases myocardial infarct size and serum CPK level. Furthermore, we show that these protective roles of SIRT4 against MI-R injury are associated with preserved mitochondrial function and reduced myocardial apoptosis. Taken together, our findings indicate that SIRT4 ameliorates MI-R injury through regulating mitochondrial function and apoptosis, and suggest that manipulating SIRT4 may be of clinical benefit in MI-R injury. © 2018 Elsevier Inc. All rights reserved.
Keywords: SIRT4 Amelioration Myocardial ischemia-reperfusion injury Mitochondrial preservation Apoptosis
1. Introduction Acute myocardial ischemia-reperfusion (MI-R) injury has detrimental effects on coronary heart disease (CHD), which is the leading cause of death worldwide [1]. MI-R injury typically occurs in patients with an acute ST-segment elevation myocardial infarction (STEMI), and the most effective therapeutic intervention for alleviating myocardial ischemic injury is myocardial reperfusion achieved through either thrombolytic therapy or primary percutaneous coronary intervention (PPCI) [2]. However, myocardial reperfusion arouses further injuries, including cardiomyocyte dysfunction, apoptosis and cell death [3]. On the other hand, mitochondria are critical targets of ischemia and subsequent reperfusion, and they have emerged as key participants and regulators of MI-R injury [4]. Following coronary occlusion, a severe reduction in blood flow generates deleterious cellular processes, especially damage to mitochondria, which result in eventual cardiomyocyte death through activating apoptosis either from mitochondrial permeability transition pore (MPTP) or via mitochondrial outer membrane permeabilization (MOMP) [5,6]. Therefore, therapeutic strategies of preventing cardiomyocyte apoptosis during
* Corresponding author. Department of Cardiology, Tangdu Hospital, the Fourth Millitary University Affiliations, 569 Xinsi Road, Xian 710038, China. E-mail address: [email protected] (H. Wang).
reperfusion may reduce reperfusion-induced cell death and improve heart function [7]. Sirtuins belong to a highly conserved family of nicotinamide adenine dinucleotide-(NAD)-dependent enzymes [8]. SIRT4 is a mitochondrial-localized member of the sirtuin family and plays key roles in a wide variety of cellular processes, such as energy metabolism, stress response and longevity [9]. SIRT4 is highly expressed in the heart and is recently implicated in cardiovascular diseases [10]. However, its physiological function in MI-R injury has not yet been established. In the present study, we uncover the protective role of SIRT4 in MI/R injury, which may relate to mitochondrial protection and reduced apoptosis of cardiomyocytes. 2. Materials and methods 2.1. Antibodies and reagents The antibodies and reagents were purchased from the following sources: SIRT4 (Abcam, ab124521), GAPDH (6C5) (Santa Cruz, sc32233), Cox IV (Cell Signaling, 4844), Cleaved caspase3 (Asp175) (Cell Signaling, 9661), Caspase3 (Cell Signaling, 9662), b-actin (C4) (Santa Cruz, sc-47778), Goat anti-rabbit IgG-HRP (Abcam, ab6721), Goat anti-mouse IgG-HRP (Abcam, ab6789), Evans blue and 2,3,5triphenyltetrazolium chloride (TTC) were purchased from Sigma.
https://doi.org/10.1016/j.bbrc.2018.05.113 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
2
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
2.2. Animals
2.6. Measurement of mitochondrial respiratory rate
All procedures involved in animal experiments were conducted in accordance with protocols and guidelines approved by the Institutional Animal Care and Use Committees of Tangdu Hospital, the Fourth Millitary University for animal welfare. Adult wild-type C57BL/6 male mice were used as animal models in this study. Generally, mice were fed and maintained under specific pathogenfree conditions, and at 12-week-old, mice were randomly divided into different groups by weight prior to experimental treatment.
The oxygen consumption of purified cardiac mitochondrial was measured as adapted from Ref. [11]. Briefly, isolated mitochondria (1 mg/ml) were initially incubated in respiration buffer composed of 120 mM KCl, 3 mM HEPES, 1 mM EGTA, 25 mM sucrose, 5 mM MgCl2, 5 mM KH2PO4, pH 7.4. Complex I respiration was measured by adding 5 mM pyruvate and 2.5 mM malate into the respiration buffer prior to the mitochondria, which was followed by 1 mM ADP addition. Alternatively, for measuring Complex II mediated oxygen consumption, 8 mM succinate, 4 mM glycerol-3-phosphate and 5 mM rotenone were added into the respiration buffer prior to the addition of mitochondria followed by 1 mM ADP addition.
2.3. Mouse model of myocardial ischemia-reperfusion The mouse model of myocardial ischemia-reperfusion (MI-R) was established as previously described [11]. Briefly, mice were anaesthetized by intraperitoneally injecting a mix solution of 50 mg/kg sodium pentobarbital and 60 mg/kg ketamine. In addition, before surgery, 200 units/kg sodium heparin was also administrated to prevent clot formation. Anaesthetized mice were placed in a supine position, and intubated with an endotracheal tube (PE-60) and effectively ventilated with 100% oxygen (0.5 L/ min) using a ventilator (HugoSachs, Model 845), which was set with conditions of a rate of 110 strokes/minute, 230-ml tidal volume and constant 37 C. The mice were then denuded and the exposed regions were sterilized with alcohol and Betadine solution. The ribcage was exposed by making a midline incision along the sternum. A thoracotomy was performed at the left of the midline using an electrocautery. The second and third ribs were cauterized to make a vertical opening, and the left coronary artery (LCA) was visualized under an Olympus SZ61 stereomicroscope. The LCA was then ligated with a 7-0 silk suture underneath the coronary artery. A small piece of PE-10 tubing was then placed along with the LCA and the 7-0 suture was tightly tied to compress the LCA and render the left ventricle ischemic for 30 min. 7-0 silk suture was then removed to reperfuse the LCA for 48 h. Eventually, the sternum and skin was closed. 2.4. Assessment of area at risk, infarct size and serum CPK release The assessments of AAR and infarct area were performed as previously documented [12]. The LCA arteries of mice were reoccluded and injected 1 ml of 1.0% Evans blue through the jugular vein to delineate the nonischemic tissue. Hearts were then excised, washed with PBS and cut into four transverse slices, which were stained for 5 min at 23 C with 1.0 ml of 1.5% TTC to determine infarct area. The final stained samples were photographed under a microscope. The left ventricular area, AAR and infarct area were measured by computerized planimetry using Image J software. The infarct area was expressed as a percentage of the AAR and left ventricular area. For analyzing the serum CPK release as an index of myocyte injury, the blood samples of mice were collected from tail vein at 6 h after operation. The serum level of CPK was determined using Creatine Kinase (CK) (CPK) kit (Catachem, V184-12) according to the manufactural instructions. 2.5. Isolation, culture and treatment of cardiomyocytes The isolation of adult mouse cardiomyocytes was performed with primary cardiomyocyte isolation kit (Pierce, 88281) according to the manufactural instructions. The final isolated cardiomyocytes were cultured in MEM. Hypoxia-reoxygenation (HR) of cultured cardiomyocytes was completed by 6-h of hypoxia (95% N, 5% CO2) and followed by 12-h reoxygenation (5% CO2) to mimic myocardial ischemia-reperfusion.
2.7. Western blotting Protocols were described as previously [13]. PVDF membranes were immersed in the enhanced chemiluminescence (GE Healthcare, RPN2209) for protein detection via GE ImageQuant LAS 4000 instrument. The band intensity was quantified using ImageJ software. 2.8. qRT-PCR Protocols were described as previously [13]. The sequences of primers were listed as follows: Sirt4 forward 50 -CGAGGGGACAAGGAGGATTT-30 , reverse 50 -GTCGGCCTGAAAGTCAATCC-30 ; Actb forward 50 -ACTGGGACGACATGGAGAAG-30 , reverse 50 GTCTCCGGAGTCCATCACAA-30 . 2.9. Statistics Statistics was analyzed with GraphPad Prism 6 software. All data are representative of at least 3 independent experiments and presented as mean ± s.d. The statistical difference was calculated by unpaired Student's t-test, unless indicated otherwise. P < 0.001, P < 0.01 and P < 0.05 indicate a statistical difference, and NS indicates no statistical difference. 3. Results 3.1. SIRT4 is downregulated in cardiomyocytes after MI-R To explore whether SIRT4 could play a possible role in MI-R injury, we first examined its expression level in cultured adult mouse cardiomyocytes subjected to treatments of 6 h of hypoxia and subsequent 12 h of reoxygenation in vitro for mimicking MI-R model [11]. Surprisingly, compared with sham treatment, the protein level of SIRT4 was decreased nearly by half (P < 0.01) in cardiomyocytes after MI-R in vitro (Fig. 1A). Consistent with this result, the transcript level of SIRT4 was also parallelly decreased (P < 0.01), although with less extent of change (Fig. 1B). To test whether this phenomenon could be extended to in vivo scenario, we next examined the expression level of SIRT4 in MI-R mice model subjected to 30 min of left ventricle (LV) ligation followed by 48 h of reperfusion. The results showed that compared with non-ischemic hearts (sham), the protein level of SIRT4 in cardiomyocytes from non-ischemic zone (NIZ) was not obviously affected (Fig. 1C, lane 1 and lane 2), whereas, the protein levels of SIRT4 in cardiomyocytes from both the border zone and ischemic zone of ischemic myocardium were significantly decreased, and with the latter being the lowest one (Fig. 1C). In addition, consistently, the transcript level of SIRT4 showed similar tendency compared with its protein level (Fig. 1D). Collectively, these results indicate that SIRT4 is downregulated in both transcript and protein levels in
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3
Fig. 1. SIRT4 is downregulated in cardiomyocytes after MI-R. (A-B) Isolated adult mouse cardiomyocytes were normally cultured (sham) or subjected to 6-h hypoxia and subsequent 12-h reoxygenation (MI-R). The protein (A) and mRNA (B) levels of SIRT4 were analyzed by Western blotting and qRT-PCR analysis, respectively. b-actin was used as a loading and reference control. Experiments were conducted at three times independently. The representative images (A, Left) and the statistical analysis of band intensity (A, Right) were shown. Data were compared with sham group. Data are mean ± s.d. n ¼ 3. **, P < 0.01. (C-D) Mice (n ¼ 5) were subjected to sham surgery or experimental MI-R. 2 days later, the protein (C) and mRNA (D) levels of SIRT4 in hearts of sham group and different regions of hearts in MI-R group were analyzed by Western blotting and qRT-PCR analysis, respectively. Samples from hearts of sham group were used as controls. b-actin was used as a loading and reference control. Experiments were conducted at three times independently. The representative images (C, Left) and the statistical analysis of band intensity (D, Right) were shown. Data were compared with sham group. Data are mean ± s.d. n ¼ 3. **, P < 0.01; *, P < 0.05; NS, not significant. NIZ, nonischemic zone; BZ, border zone; IZ, ischemic zone.
cardiomyocytes after MI-R.
3.2. SIRT4 ameliorates MI-R injury In order to investigate the potential function of SIRT4 in MI-R injury in vivo, we overexpressed SIRT4 in mice hearts by locally delivering adenovirus (Ad-SIRT4), mice were allowed to complete SIRT4 overexpression for 3 days and then subjected to experimental MI-R. As shown, compared with Ad-vector group, the hearts of Ad-SIRT4 group displayed smaller infarct size as assessed by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining (Fig. 2A). The ratio of area at risk (AAR, delineated by Evans blue) to LV area was the same in both groups, but the ratios of infarct area (IA, delineated by TTC) to AAR and IA to LV area were decreased by 46% and 48%, respectively, in Ad-SIRT4 group compared with those of Ad-vector group (Fig. 2B). These results reveal a protective function of SIRT4 in MI-R injury in vivo. To strengthen this notion, we measured the level of serum creatine phosphokinase (CPK), which is an index of myocyte injury [14]. As expected, the CPK level was not changed in sham group, but was sharply increased after MI-R injury, indicating a severe myocyte injury (Fig. 2C). However, in mice with MI-R injury, the increased level of CPK was
significantly attenuated in Ad-SIRT4 group, as compared with Advector group (Fig. 2C). Therefore, these lines of evidence indicate that SIRT4 overexpression provides protection against MI-R injury. Oppositely, we examine whether SIRT4 inhibition could deteriorate MI-R injury. We knocked down SIRT4 expression by locally delivering the small interfering RNA (siRNA) specific targeting SIRT4 (siSIRT4) into the hearts. In contrast to results obtained from SIRT4 overexpression, results showed that compared with siCtrl group, greater infarct size was found in siSIRT4 group (Fig. 2D). As evaluated, the ratio of AAR to LV area was the same in both groups, but the ratios of IA to AAR and IA to LV area were increased by 42% and 46%, respectively, in siSIRT4 group compared with those of siCtrl group (Fig. 2E). Moreover, the elevated level of serum CPK induced by MI-R injury was further enhanced in siSIRT4 group, as compared with those of siCtrl group (Fig. 2F). Thus, these results reveal that SIRT4 inhibition deteriorates the severity of MI-R injury. Taken together, SIRT4 ameliorates MI-R injury in vivo.
3.3. SIRT4 preserves mitochondrial function in vivo after MI-R To further investigate whether SIRT4 could preserve mitochondrial function after MI-R in vivo, we checked the respiratory
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
4
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Fig. 2. SIRT4 ameliorates MI/R injury. (AeF) Mice hearts were transfected in vivo with adenovirus expressing vector control (Ad-vector) or SIRT4 (Ad-SIRT4) (AeC), or with siRNA targeting luciferase (siCtrl) or SIRT4 (siSIRT4) (DeF). 3 days later, mice were subjected to sham surgery or experimental MI-R. Each group contained 5 mice. After 2 days, mice were analyzed as follows. (A and D) Representative images of mid-myocardial cross sections of Evans blue and TTC-stained hearts in MI-R groups (upper) and representative Western blotting band of SIRT4 (below). The nonischemic area is indicated by dark blue, the area at risk (AAR) by red and the infarct area (IA) by white. b-actin was used as a loading control. (B and E) The quantification of infarct size in MI-R groups. AAR/LV, ratio of AAR to left ventricular area; IA/AAR, ratio of infarct area to AAR; IA/LV, ratio of infarct area to left ventricular area. (C and F) Levels of serum creatine phosphokinase (CPK) from sham and MI-R groups. Data were compared with Ad-vector group. Data are mean ± s.d. n ¼ 3. **, P < 0.01; *, P < 0.05; NS, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
rate in cardiac mitochondria isolated from mice hearts subjected to sham surgery or MI-R. Compared with sham group, the isolated mitochondria in MI-R group were found to have a 73% decrease in the rate of oxygen consumption, however SIRT4 overexpression (Fig. 3A) considerably rescued the decreased oxygen consumption rate, as measured with complex I substrates, pyruvate and malate (Fig. 3B). The complex II efficiency was also determined, and similar results were obtained, as evaluated with complex II substrates, succinate and G3P (Fig. 3C). Hence, SIRT4 overexpression preserves mitochondrial function after MI-R in vivo. We next test whether SIRT4 inhibition could exhaust mitochondrial function after MI-R in vivo. As shown, SIRT4 inhibition mediated by siRNA (Fig. 3D) further significantly reduced the decreased rate of oxygen consumption in isolated mitochondria caused by MI-R, as measured by efficiencies of complex I (Fig. 3E) and complex II (Fig. 3F). These findings indicate an exhausted mitochondrial function elicited by SIRT4 inhibition. Taken together, SIRT4 serves to preserve mitochondrial function after MI-R in vivo, which may contribute to its protective role in MI-R injury. 3.4. Sirt4 reduces cardiomyocyte apoptosis after MI-R Mitochondria dysfunction is a key executioner for triggering apoptosis [15], this prompted us to explore the role of SIRT4 in cardiomyocyte apoptosis induced by MI-R. We found that compared with sham group, MI-R strikingly induced the apoptosis
of cardiomyocytes, and reversely, the overexpression of SIRT4 reduced the percentage of apoptotic cardiomyocytes caused by MIR, as determined by TUNEL staining (Fig. 4A and B). Consistent with this result, the increased protein level of cleaved caspase3, one indicator of apoptosis, in isolated cardiomyocytes caused by MI-R was also largely attenuated by SIRT4 overexpression (Fig. 4C). And vice versa, SIRT4 inhibition further increased the percentage of apoptotic cardiomyocytes caused by MI-R (Fig. 4D and E). Furthermore, SIRT4 inhibition also further increased the elevated protein level of cleaved caspase3 caused by MI-R. Thus, SIRT4 reduces cardiomyocyte apoptosis after MI-R in vivo, and this effect at least in part accounts for its cytoprotective function after MI-R. 4. Disscussion Different from SIRT1 and SIRT3, not much is uncovered about the physiology of SIRT4. In this study, we in the first time investigated the physiology role of SIRT4 in MI-R injury and explored the underlying mechanisms. Initially, through utilizing MI-R models in vitro and in vivo, we noticed that the expression level of SIRT4 was downregulated in cardiomyocytes after MI-R. Following functional studies revealed an ameliorating function of SIRT4 for MI-R injury in vivo. Mechanistically, we showed that this protective function of SIRT4 against MI-R injury may involve preserved mitochondrial function and reduced cardiomyocyte apoptosis. Our evidence illustrated that the transcript level of SIRT4 was
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
5
Fig. 3. SIRT4 preserves mitochondrial function in vivo after MI-R. (A-F) Mice hearts were transfected in vivo with Ad-vector or Ad-SIRT4 (AeC), or with siCtrl or siSIRT4 (DeF). 3 days later, mice were subjected to mice were subjected to sham surgery or experimental MI-R. Each group contained 5 mice. After 2 days, the mitochondria were then isolated from cardiomyocytes. (A and D) The protein levels of SIRT4, GAPDH and Cox-IV were analyzed by Western blotting. The representative images were shown. (B and E) The efficiency of mitochondrial complex I was determined by using pyruvate and malate as substrates. Each group contained mitochondria isolated from 5 mice. Data were compared with as indicated. Data are mean ± s.d. n ¼ 3. **, P < 0.01. (C and F) The efficiency of mitochondrial complex II was determined by using succinate and G3P as substrates and inhibiting complex I with rotenone. Each group contained mitochondria isolated from 5 mice. Data were compared with as indicated. Data are mean ± s.d. n ¼ 3. **, P < 0.01.
parallelly increased along with its protein level in cardiomyocyte. This suggests a transcriptional activation of SIRT4 in response to MI-R. However, whether its enzymatic activity is affected by MI-R is unknown based on our available data. At present, little is known about the transcriptional control of SIRT4. In this regard, among the sirtuin family members, the best-described is SIRT1 [16]. The expression level of SIRT1 varies according to various physiological conditions. For instance, its expression can be transcriptionally induced under low cellular energy status, including nutrient starvation [17]. Transcription factors including FOXO1, CREB, CHREBP and PPARs are supposed to regulate SIRT1 expression in response to these stimuli [16]. Noticeably, in our study, we found that the expression of SIRT4 was not decreased in the non-ischemic zone, but were significantly decreased in the border zone and ischemic zone of ischemic myocardium, and with its expression level the lowest one in ischemic zone. These strongly imply that the expression level of SIRT4 is reversely correlated with ischemic extent. In ischemic myocardium caused by MI-R, the energy supply is impaired, leading to a low energy status [18]. Therefore, we suppose it is very possible that the reduced SIRT4 expression may be due to regulation by certain transcriptional factors as mentioned above in response to low energy status engendered by MI-R. To clearly clarify this, further studies are needed to elucidate which
transcriptional factor(s) is responsible for activating SIRT4 transcription under MI-R condition. SIRT4 is highly expressed in the heart. Recently, it has been shown that SIRT4 is involved in the pathogenesis of pathological cardiac hypertrophy through regulating the production of reactive oxygen species (ROS) [10], suggesting it may participate in other cardiovascular diseases. We revealed that SIRT4 was able to ameliorate MI/R injury in vivo, expanding its role in another cardiovascular disease. SIRT4 is a mitochondrial-localized sirtuin and is functionally versatile [19]. We next found that SIRT4 preserved mitochondrial function and reduced cardiomyocyte apoptosis, therefore providing two possible mechanisms which may account for its protective function against MI/R injury. The preservation of mitochondrial function is an important aspect in myocardial cytoprotection [20]. The mitochondria are central sites for not only cellular energy production but also for controlling cell death [21]. Maintaining the homeostasis of oxidative phosphorylation within mitochondria to prevent cardiomyocyte death during ischemic injury has long been viewed as a critical event after myocardial infarction [22]. We discovered that mitochondria overexpressing SIRT4 and isolated from mice subjected to MI/R displayed preserved mitochondrial function as measured by increased complex I and II efficiency, and vice versa,
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
6
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Fig. 4. Sirt4 reduces cardiomyocyte apoptosis after MI/R. (A-F) Mice hearts were transfected in vivo with Ad-vector or Ad-SIRT4 (AeC), or with siCtrl or siSIRT4 (DeE). 3 days later, mice were subjected to sham surgery or experimental MI-R. Each group contained 5 mice. After 2 days, mice were analyzed as follows. (A and D) Representative images of TUNEL-stained heart sections from 4 groups. Apoptotic nuclei were identified by TUNEL staining (green) and total nuclei by DAPI counterstaining (blue). (B and E) Quantitative analysis of apoptotic nuclei. TUNEL-positive nuclei are expressed as a percentage of the total number of nuclei. Data were compared with Ad-vector group. Data are mean ± s.d. n ¼ 5. **, P < 0.01; NS, not significant. (C and F) Cardiomyocytes were isolated and analyzed by Western blotting to detect levels of SIRT4, Cleaved caspase3 and Caspase3. b-actin was used as a loading control. The representative images (left) and the statistical analysis of relative intensity of Cleaved caspase3/Caspase3 (right) were shown. Data were compared with Ad-vector group. Data are mean ± s.d. n ¼ 5. **, P < 0.01; NS, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
SIRT4 deficiency exhausted mitochondrial function. These lines of evidence suggest a role of preserving the mitochondrial function in the observed cytoprotection. Detection of mitochondrial structure in this scenario may provide useful evidence to further demonstrate whether SIRT4 preserves mitochondrial function after MI/R. We speculate that one possible mechanism for SIRT4 protective role in mitochondrial function may lie in its ability to modulate cellular respiration during reperfusion. It has been shown that the suppressed mitochondrial respiration protects against MI-R injury by limiting ROS production and mitochondrial uncoupling degree, which lead to decreased infarct size and preserved mitochondrial function [23]. However, to what extent the ROS production [10] and mitochondrial uncoupling [24] regulated by SIRT4 contribute to its role in MI/R need further interrogations. The protective role of SIRT4 was further reinforced by the decreased cleaved caspase3 and a decrease number of apoptotic cardiomyocytes. These suggest that SIRT4 is capable of inhibiting the progression of apoptosis after MI-R injury, at any rate, which we believe is associated with its function in preserving mitochondrial function. Nevertheless, direct evidence is required to better understand their connections. More importantly, deconvoluting the context-dependent roles of SIRT4 related to mitochondrial function and cell apoptosis may shed light on its mechanism of action in MIR. In conclusion, our study uncovers the protective role of SIRT4 in MI-R, and relates this effect to mitochondrial protection and reduced apoptosis. Thus, we propose here that enhancing the expression of SIRT4 in cardiomyocytes might be beneficial for reducing cardiomyocyte apoptosis and alleviating MI-R injury in clinic practice.
Disclosure of conflict of interest None. Acknowledgments This research was supported by the fund of Shaanxi science and technology support project (2017JCYJ007). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.113. References [1] M. Nichols, N. Townsend, P. Scarborough, M. Rayner, Cardiovascular disease in Europe: epidemiological update, Eur. Heart J. 34 (2013) 3028e3034. [2] D.J. Hausenloy, D.M. Yellon, Myocardial ischemia-reperfusion injury: a neglected therapeutic target, J. Clin. Invest. 123 (2013) 92e100. [3] R.A. Gottlieb, Cell death pathways in acute ischemia/reperfusion injury, J. Cardiovasc. Pharmacol. Therapeut. 16 (2011) 233e238. [4] E.J. Lesnefsky, Q. Chen, B. Tandler, C.L. Hoppel, Mitochondrial dysfunction and myocardial ischemia-reperfusion: implications for novel therapies, Annu. Rev. Pharmacol. Toxicol. 57 (2017) 535e565. [5] V. Borutaite, A. Jekabsone, R. Morkuniene, G.C. Brown, Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia, J. Mol. Cell. Cardiol. 35 (2003) 357e366. [6] D.A. Kubli, A.B. Gustafsson, Mitochondria and mitophagy: the yin and yang of cell death control, Circ. Res. 111 (2012) 1208e1221. [7] A. Cattelan, G. Ceolotto, S. Bova, M. Albiero, M. Kuppusamy, S. De Martin, A. Semplicini, G.P. Fadini, S.V. de Kreutzenberg, A. Avogaro, NAD(þ)-dependent SIRT1 deactivation has a key role on ischemia-reperfusion-induced apoptosis, Vasc. Pharmacol. 70 (2015) 35e44.
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 [8] M.C. Haigis, D.A. Sinclair, Mammalian sirtuins: biological insights and disease relevance, Annu. Rev. Pathol. 5 (2010) 253e295. [9] A. Csibi, S.M. Fendt, C. Li, G. Poulogiannis, A.Y. Choo, D.J. Chapski, S.M. Jeong, J.M. Dempsey, A. Parkhitko, T. Morrison, E.P. Henske, M.C. Haigis, L.C. Cantley, G. Stephanopoulos, J. Yu, J. Blenis, The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4, Cell 153 (2013) 840e854. [10] Y.X. Luo, X. Tang, X.Z. An, X.M. Xie, X.F. Chen, X. Zhao, D.L. Hao, H.Z. Chen, D.P. Liu, SIRT4 accelerates Ang II-induced pathological cardiac hypertrophy by inhibiting manganese superoxide dismutase activity, Eur. Heart J. 38 (2017) 1389e1398. [11] J.W. Elrod, J.W. Calvert, J. Morrison, J.E. Doeller, D.W. Kraus, L. Tao, X. Jiao, R. Scalia, L. Kiss, C. Szabo, H. Kimura, C.W. Chow, D.J. Lefer, Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 15560e15565. [12] R. Shibata, K. Sato, D.R. Pimentel, Y. Takemura, S. Kihara, K. Ohashi, T. Funahashi, N. Ouchi, K. Walsh, Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms, Nat. Med. 11 (2005) 1096e1103. [13] S. Gao, X. Qi, J. Li, L. Sang, Upregulated KAT7 in synovial fibroblasts promotes Th17 cell differentiation and infiltration in rheumatoid arthritis, Biochem. Biophys. Res. Commun. 489 (2017) 235e241. [14] M.F. Corsten, R. Dennert, S. Jochems, T. Kuznetsova, Y. Devaux, L. Hofstra, D.R. Wagner, J.A. Staessen, S. Heymans, B. Schroen, Circulating MicroRNA208b and MicroRNA-499 reflect myocardial damage in cardiovascular disease, Circ. Cardiovasc. Genet. 3 (2010) 499e506.
7
[15] A. Kasahara, L. Scorrano, Mitochondria: from cell death executioners to regulators of cell differentiation, Trends Cell Biol. 24 (2014) 761e770. [16] R.H. Houtkooper, E. Pirinen, J. Auwerx, Sirtuins as regulators of metabolism and healthspan, Nature reviews, Mol. Cell Biol. 13 (2012) 225e238. [17] S. Nemoto, M.M. Fergusson, T. Finkel, Nutrient availability regulates SIRT1 through a forkhead-dependent pathway, Science 306 (2004) 2105e2108. [18] L.M. Buja, Myocardial ischemia and reperfusion injury, Cardiovasc. Pathol. : The Official Journal of the Society for Cardiovascular Pathology 14 (2005) 170e175. [19] S. Kumar, D.B. Lombard, For certain, SIRT4 activities!, Trends Biochem. Sci. 42 (2017) 499e501. [20] E. Murphy, C. Steenbergen, Preconditioning: the mitochondrial connection, Annu. Rev. Physiol. 69 (2007) 51e67. [21] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99e163. [22] R.B. Jennings, C.E. Ganote, Structural changes in myocardium during acute ischemia, Circ. Res. 35 (Suppl 3) (1974) 156e172. [23] Q. Chen, A.K. Camara, D.F. Stowe, C.L. Hoppel, E.J. Lesnefsky, Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion, American journal of physiology, Cell Physiol. 292 (2007) C137eC147. [24] A. Lang, R. Anand, S. Altinoluk-Hambuchen, H. Ezzahoini, A. Stefanski, A. Iram, L. Bergmann, J. Urbach, P. Bohler, J. Hansel, M. Franke, K. Stuhler, J. Krutmann, J. Scheller, B. Stork, A.S. Reichert, R.P. Piekorz, SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy, Aging 9 (2017) 2163e2189.
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis Guangwei Zeng, Hui Liu, Haiyan Wang* Department of Cardiology, Tangdu Hospital, The Fourth Millitary University, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2018 Accepted 16 May 2018 Available online xxx
Apoptosis and mitochondria dysfunction are key contributors to myocardial ischemia-reperfusion (MI-R) injury. SIRT4, a mitochondrial-localized sirtuin, controls cellular energy metabolism and stress response, and is abundantly present in the heart, however, its role in MI-R injury is not clear. In the current study, we demonstrate that SIRT4 is downregulated in cardiomyocytes both in vitro and in vivo models after MI-R. Functionally, SIRT4 overexpression decreases myocardial infarct size and serum creatine phosphokinase (CPK) level, and vice versa, SIRT4 depletion by siRNA increases myocardial infarct size and serum CPK level. Furthermore, we show that these protective roles of SIRT4 against MI-R injury are associated with preserved mitochondrial function and reduced myocardial apoptosis. Taken together, our findings indicate that SIRT4 ameliorates MI-R injury through regulating mitochondrial function and apoptosis, and suggest that manipulating SIRT4 may be of clinical benefit in MI-R injury. © 2018 Elsevier Inc. All rights reserved.
Keywords: SIRT4 Amelioration Myocardial ischemia-reperfusion injury Mitochondrial preservation Apoptosis
1. Introduction Acute myocardial ischemia-reperfusion (MI-R) injury has detrimental effects on coronary heart disease (CHD), which is the leading cause of death worldwide [1]. MI-R injury typically occurs in patients with an acute ST-segment elevation myocardial infarction (STEMI), and the most effective therapeutic intervention for alleviating myocardial ischemic injury is myocardial reperfusion achieved through either thrombolytic therapy or primary percutaneous coronary intervention (PPCI) [2]. However, myocardial reperfusion arouses further injuries, including cardiomyocyte dysfunction, apoptosis and cell death [3]. On the other hand, mitochondria are critical targets of ischemia and subsequent reperfusion, and they have emerged as key participants and regulators of MI-R injury [4]. Following coronary occlusion, a severe reduction in blood flow generates deleterious cellular processes, especially damage to mitochondria, which result in eventual cardiomyocyte death through activating apoptosis either from mitochondrial permeability transition pore (MPTP) or via mitochondrial outer membrane permeabilization (MOMP) [5,6]. Therefore, therapeutic strategies of preventing cardiomyocyte apoptosis during
* Corresponding author. Department of Cardiology, Tangdu Hospital, the Fourth Millitary University Affiliations, 569 Xinsi Road, Xian 710038, China. E-mail address: [email protected] (H. Wang).
reperfusion may reduce reperfusion-induced cell death and improve heart function [7]. Sirtuins belong to a highly conserved family of nicotinamide adenine dinucleotide-(NAD)-dependent enzymes [8]. SIRT4 is a mitochondrial-localized member of the sirtuin family and plays key roles in a wide variety of cellular processes, such as energy metabolism, stress response and longevity [9]. SIRT4 is highly expressed in the heart and is recently implicated in cardiovascular diseases [10]. However, its physiological function in MI-R injury has not yet been established. In the present study, we uncover the protective role of SIRT4 in MI/R injury, which may relate to mitochondrial protection and reduced apoptosis of cardiomyocytes. 2. Materials and methods 2.1. Antibodies and reagents The antibodies and reagents were purchased from the following sources: SIRT4 (Abcam, ab124521), GAPDH (6C5) (Santa Cruz, sc32233), Cox IV (Cell Signaling, 4844), Cleaved caspase3 (Asp175) (Cell Signaling, 9661), Caspase3 (Cell Signaling, 9662), b-actin (C4) (Santa Cruz, sc-47778), Goat anti-rabbit IgG-HRP (Abcam, ab6721), Goat anti-mouse IgG-HRP (Abcam, ab6789), Evans blue and 2,3,5triphenyltetrazolium chloride (TTC) were purchased from Sigma.
https://doi.org/10.1016/j.bbrc.2018.05.113 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
2
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
2.2. Animals
2.6. Measurement of mitochondrial respiratory rate
All procedures involved in animal experiments were conducted in accordance with protocols and guidelines approved by the Institutional Animal Care and Use Committees of Tangdu Hospital, the Fourth Millitary University for animal welfare. Adult wild-type C57BL/6 male mice were used as animal models in this study. Generally, mice were fed and maintained under specific pathogenfree conditions, and at 12-week-old, mice were randomly divided into different groups by weight prior to experimental treatment.
The oxygen consumption of purified cardiac mitochondrial was measured as adapted from Ref. [11]. Briefly, isolated mitochondria (1 mg/ml) were initially incubated in respiration buffer composed of 120 mM KCl, 3 mM HEPES, 1 mM EGTA, 25 mM sucrose, 5 mM MgCl2, 5 mM KH2PO4, pH 7.4. Complex I respiration was measured by adding 5 mM pyruvate and 2.5 mM malate into the respiration buffer prior to the mitochondria, which was followed by 1 mM ADP addition. Alternatively, for measuring Complex II mediated oxygen consumption, 8 mM succinate, 4 mM glycerol-3-phosphate and 5 mM rotenone were added into the respiration buffer prior to the addition of mitochondria followed by 1 mM ADP addition.
2.3. Mouse model of myocardial ischemia-reperfusion The mouse model of myocardial ischemia-reperfusion (MI-R) was established as previously described [11]. Briefly, mice were anaesthetized by intraperitoneally injecting a mix solution of 50 mg/kg sodium pentobarbital and 60 mg/kg ketamine. In addition, before surgery, 200 units/kg sodium heparin was also administrated to prevent clot formation. Anaesthetized mice were placed in a supine position, and intubated with an endotracheal tube (PE-60) and effectively ventilated with 100% oxygen (0.5 L/ min) using a ventilator (HugoSachs, Model 845), which was set with conditions of a rate of 110 strokes/minute, 230-ml tidal volume and constant 37 C. The mice were then denuded and the exposed regions were sterilized with alcohol and Betadine solution. The ribcage was exposed by making a midline incision along the sternum. A thoracotomy was performed at the left of the midline using an electrocautery. The second and third ribs were cauterized to make a vertical opening, and the left coronary artery (LCA) was visualized under an Olympus SZ61 stereomicroscope. The LCA was then ligated with a 7-0 silk suture underneath the coronary artery. A small piece of PE-10 tubing was then placed along with the LCA and the 7-0 suture was tightly tied to compress the LCA and render the left ventricle ischemic for 30 min. 7-0 silk suture was then removed to reperfuse the LCA for 48 h. Eventually, the sternum and skin was closed. 2.4. Assessment of area at risk, infarct size and serum CPK release The assessments of AAR and infarct area were performed as previously documented [12]. The LCA arteries of mice were reoccluded and injected 1 ml of 1.0% Evans blue through the jugular vein to delineate the nonischemic tissue. Hearts were then excised, washed with PBS and cut into four transverse slices, which were stained for 5 min at 23 C with 1.0 ml of 1.5% TTC to determine infarct area. The final stained samples were photographed under a microscope. The left ventricular area, AAR and infarct area were measured by computerized planimetry using Image J software. The infarct area was expressed as a percentage of the AAR and left ventricular area. For analyzing the serum CPK release as an index of myocyte injury, the blood samples of mice were collected from tail vein at 6 h after operation. The serum level of CPK was determined using Creatine Kinase (CK) (CPK) kit (Catachem, V184-12) according to the manufactural instructions. 2.5. Isolation, culture and treatment of cardiomyocytes The isolation of adult mouse cardiomyocytes was performed with primary cardiomyocyte isolation kit (Pierce, 88281) according to the manufactural instructions. The final isolated cardiomyocytes were cultured in MEM. Hypoxia-reoxygenation (HR) of cultured cardiomyocytes was completed by 6-h of hypoxia (95% N, 5% CO2) and followed by 12-h reoxygenation (5% CO2) to mimic myocardial ischemia-reperfusion.
2.7. Western blotting Protocols were described as previously [13]. PVDF membranes were immersed in the enhanced chemiluminescence (GE Healthcare, RPN2209) for protein detection via GE ImageQuant LAS 4000 instrument. The band intensity was quantified using ImageJ software. 2.8. qRT-PCR Protocols were described as previously [13]. The sequences of primers were listed as follows: Sirt4 forward 50 -CGAGGGGACAAGGAGGATTT-30 , reverse 50 -GTCGGCCTGAAAGTCAATCC-30 ; Actb forward 50 -ACTGGGACGACATGGAGAAG-30 , reverse 50 GTCTCCGGAGTCCATCACAA-30 . 2.9. Statistics Statistics was analyzed with GraphPad Prism 6 software. All data are representative of at least 3 independent experiments and presented as mean ± s.d. The statistical difference was calculated by unpaired Student's t-test, unless indicated otherwise. P < 0.001, P < 0.01 and P < 0.05 indicate a statistical difference, and NS indicates no statistical difference. 3. Results 3.1. SIRT4 is downregulated in cardiomyocytes after MI-R To explore whether SIRT4 could play a possible role in MI-R injury, we first examined its expression level in cultured adult mouse cardiomyocytes subjected to treatments of 6 h of hypoxia and subsequent 12 h of reoxygenation in vitro for mimicking MI-R model [11]. Surprisingly, compared with sham treatment, the protein level of SIRT4 was decreased nearly by half (P < 0.01) in cardiomyocytes after MI-R in vitro (Fig. 1A). Consistent with this result, the transcript level of SIRT4 was also parallelly decreased (P < 0.01), although with less extent of change (Fig. 1B). To test whether this phenomenon could be extended to in vivo scenario, we next examined the expression level of SIRT4 in MI-R mice model subjected to 30 min of left ventricle (LV) ligation followed by 48 h of reperfusion. The results showed that compared with non-ischemic hearts (sham), the protein level of SIRT4 in cardiomyocytes from non-ischemic zone (NIZ) was not obviously affected (Fig. 1C, lane 1 and lane 2), whereas, the protein levels of SIRT4 in cardiomyocytes from both the border zone and ischemic zone of ischemic myocardium were significantly decreased, and with the latter being the lowest one (Fig. 1C). In addition, consistently, the transcript level of SIRT4 showed similar tendency compared with its protein level (Fig. 1D). Collectively, these results indicate that SIRT4 is downregulated in both transcript and protein levels in
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3
Fig. 1. SIRT4 is downregulated in cardiomyocytes after MI-R. (A-B) Isolated adult mouse cardiomyocytes were normally cultured (sham) or subjected to 6-h hypoxia and subsequent 12-h reoxygenation (MI-R). The protein (A) and mRNA (B) levels of SIRT4 were analyzed by Western blotting and qRT-PCR analysis, respectively. b-actin was used as a loading and reference control. Experiments were conducted at three times independently. The representative images (A, Left) and the statistical analysis of band intensity (A, Right) were shown. Data were compared with sham group. Data are mean ± s.d. n ¼ 3. **, P < 0.01. (C-D) Mice (n ¼ 5) were subjected to sham surgery or experimental MI-R. 2 days later, the protein (C) and mRNA (D) levels of SIRT4 in hearts of sham group and different regions of hearts in MI-R group were analyzed by Western blotting and qRT-PCR analysis, respectively. Samples from hearts of sham group were used as controls. b-actin was used as a loading and reference control. Experiments were conducted at three times independently. The representative images (C, Left) and the statistical analysis of band intensity (D, Right) were shown. Data were compared with sham group. Data are mean ± s.d. n ¼ 3. **, P < 0.01; *, P < 0.05; NS, not significant. NIZ, nonischemic zone; BZ, border zone; IZ, ischemic zone.
cardiomyocytes after MI-R.
3.2. SIRT4 ameliorates MI-R injury In order to investigate the potential function of SIRT4 in MI-R injury in vivo, we overexpressed SIRT4 in mice hearts by locally delivering adenovirus (Ad-SIRT4), mice were allowed to complete SIRT4 overexpression for 3 days and then subjected to experimental MI-R. As shown, compared with Ad-vector group, the hearts of Ad-SIRT4 group displayed smaller infarct size as assessed by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining (Fig. 2A). The ratio of area at risk (AAR, delineated by Evans blue) to LV area was the same in both groups, but the ratios of infarct area (IA, delineated by TTC) to AAR and IA to LV area were decreased by 46% and 48%, respectively, in Ad-SIRT4 group compared with those of Ad-vector group (Fig. 2B). These results reveal a protective function of SIRT4 in MI-R injury in vivo. To strengthen this notion, we measured the level of serum creatine phosphokinase (CPK), which is an index of myocyte injury [14]. As expected, the CPK level was not changed in sham group, but was sharply increased after MI-R injury, indicating a severe myocyte injury (Fig. 2C). However, in mice with MI-R injury, the increased level of CPK was
significantly attenuated in Ad-SIRT4 group, as compared with Advector group (Fig. 2C). Therefore, these lines of evidence indicate that SIRT4 overexpression provides protection against MI-R injury. Oppositely, we examine whether SIRT4 inhibition could deteriorate MI-R injury. We knocked down SIRT4 expression by locally delivering the small interfering RNA (siRNA) specific targeting SIRT4 (siSIRT4) into the hearts. In contrast to results obtained from SIRT4 overexpression, results showed that compared with siCtrl group, greater infarct size was found in siSIRT4 group (Fig. 2D). As evaluated, the ratio of AAR to LV area was the same in both groups, but the ratios of IA to AAR and IA to LV area were increased by 42% and 46%, respectively, in siSIRT4 group compared with those of siCtrl group (Fig. 2E). Moreover, the elevated level of serum CPK induced by MI-R injury was further enhanced in siSIRT4 group, as compared with those of siCtrl group (Fig. 2F). Thus, these results reveal that SIRT4 inhibition deteriorates the severity of MI-R injury. Taken together, SIRT4 ameliorates MI-R injury in vivo.
3.3. SIRT4 preserves mitochondrial function in vivo after MI-R To further investigate whether SIRT4 could preserve mitochondrial function after MI-R in vivo, we checked the respiratory
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
4
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Fig. 2. SIRT4 ameliorates MI/R injury. (AeF) Mice hearts were transfected in vivo with adenovirus expressing vector control (Ad-vector) or SIRT4 (Ad-SIRT4) (AeC), or with siRNA targeting luciferase (siCtrl) or SIRT4 (siSIRT4) (DeF). 3 days later, mice were subjected to sham surgery or experimental MI-R. Each group contained 5 mice. After 2 days, mice were analyzed as follows. (A and D) Representative images of mid-myocardial cross sections of Evans blue and TTC-stained hearts in MI-R groups (upper) and representative Western blotting band of SIRT4 (below). The nonischemic area is indicated by dark blue, the area at risk (AAR) by red and the infarct area (IA) by white. b-actin was used as a loading control. (B and E) The quantification of infarct size in MI-R groups. AAR/LV, ratio of AAR to left ventricular area; IA/AAR, ratio of infarct area to AAR; IA/LV, ratio of infarct area to left ventricular area. (C and F) Levels of serum creatine phosphokinase (CPK) from sham and MI-R groups. Data were compared with Ad-vector group. Data are mean ± s.d. n ¼ 3. **, P < 0.01; *, P < 0.05; NS, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
rate in cardiac mitochondria isolated from mice hearts subjected to sham surgery or MI-R. Compared with sham group, the isolated mitochondria in MI-R group were found to have a 73% decrease in the rate of oxygen consumption, however SIRT4 overexpression (Fig. 3A) considerably rescued the decreased oxygen consumption rate, as measured with complex I substrates, pyruvate and malate (Fig. 3B). The complex II efficiency was also determined, and similar results were obtained, as evaluated with complex II substrates, succinate and G3P (Fig. 3C). Hence, SIRT4 overexpression preserves mitochondrial function after MI-R in vivo. We next test whether SIRT4 inhibition could exhaust mitochondrial function after MI-R in vivo. As shown, SIRT4 inhibition mediated by siRNA (Fig. 3D) further significantly reduced the decreased rate of oxygen consumption in isolated mitochondria caused by MI-R, as measured by efficiencies of complex I (Fig. 3E) and complex II (Fig. 3F). These findings indicate an exhausted mitochondrial function elicited by SIRT4 inhibition. Taken together, SIRT4 serves to preserve mitochondrial function after MI-R in vivo, which may contribute to its protective role in MI-R injury. 3.4. Sirt4 reduces cardiomyocyte apoptosis after MI-R Mitochondria dysfunction is a key executioner for triggering apoptosis [15], this prompted us to explore the role of SIRT4 in cardiomyocyte apoptosis induced by MI-R. We found that compared with sham group, MI-R strikingly induced the apoptosis
of cardiomyocytes, and reversely, the overexpression of SIRT4 reduced the percentage of apoptotic cardiomyocytes caused by MIR, as determined by TUNEL staining (Fig. 4A and B). Consistent with this result, the increased protein level of cleaved caspase3, one indicator of apoptosis, in isolated cardiomyocytes caused by MI-R was also largely attenuated by SIRT4 overexpression (Fig. 4C). And vice versa, SIRT4 inhibition further increased the percentage of apoptotic cardiomyocytes caused by MI-R (Fig. 4D and E). Furthermore, SIRT4 inhibition also further increased the elevated protein level of cleaved caspase3 caused by MI-R. Thus, SIRT4 reduces cardiomyocyte apoptosis after MI-R in vivo, and this effect at least in part accounts for its cytoprotective function after MI-R. 4. Disscussion Different from SIRT1 and SIRT3, not much is uncovered about the physiology of SIRT4. In this study, we in the first time investigated the physiology role of SIRT4 in MI-R injury and explored the underlying mechanisms. Initially, through utilizing MI-R models in vitro and in vivo, we noticed that the expression level of SIRT4 was downregulated in cardiomyocytes after MI-R. Following functional studies revealed an ameliorating function of SIRT4 for MI-R injury in vivo. Mechanistically, we showed that this protective function of SIRT4 against MI-R injury may involve preserved mitochondrial function and reduced cardiomyocyte apoptosis. Our evidence illustrated that the transcript level of SIRT4 was
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
5
Fig. 3. SIRT4 preserves mitochondrial function in vivo after MI-R. (A-F) Mice hearts were transfected in vivo with Ad-vector or Ad-SIRT4 (AeC), or with siCtrl or siSIRT4 (DeF). 3 days later, mice were subjected to mice were subjected to sham surgery or experimental MI-R. Each group contained 5 mice. After 2 days, the mitochondria were then isolated from cardiomyocytes. (A and D) The protein levels of SIRT4, GAPDH and Cox-IV were analyzed by Western blotting. The representative images were shown. (B and E) The efficiency of mitochondrial complex I was determined by using pyruvate and malate as substrates. Each group contained mitochondria isolated from 5 mice. Data were compared with as indicated. Data are mean ± s.d. n ¼ 3. **, P < 0.01. (C and F) The efficiency of mitochondrial complex II was determined by using succinate and G3P as substrates and inhibiting complex I with rotenone. Each group contained mitochondria isolated from 5 mice. Data were compared with as indicated. Data are mean ± s.d. n ¼ 3. **, P < 0.01.
parallelly increased along with its protein level in cardiomyocyte. This suggests a transcriptional activation of SIRT4 in response to MI-R. However, whether its enzymatic activity is affected by MI-R is unknown based on our available data. At present, little is known about the transcriptional control of SIRT4. In this regard, among the sirtuin family members, the best-described is SIRT1 [16]. The expression level of SIRT1 varies according to various physiological conditions. For instance, its expression can be transcriptionally induced under low cellular energy status, including nutrient starvation [17]. Transcription factors including FOXO1, CREB, CHREBP and PPARs are supposed to regulate SIRT1 expression in response to these stimuli [16]. Noticeably, in our study, we found that the expression of SIRT4 was not decreased in the non-ischemic zone, but were significantly decreased in the border zone and ischemic zone of ischemic myocardium, and with its expression level the lowest one in ischemic zone. These strongly imply that the expression level of SIRT4 is reversely correlated with ischemic extent. In ischemic myocardium caused by MI-R, the energy supply is impaired, leading to a low energy status [18]. Therefore, we suppose it is very possible that the reduced SIRT4 expression may be due to regulation by certain transcriptional factors as mentioned above in response to low energy status engendered by MI-R. To clearly clarify this, further studies are needed to elucidate which
transcriptional factor(s) is responsible for activating SIRT4 transcription under MI-R condition. SIRT4 is highly expressed in the heart. Recently, it has been shown that SIRT4 is involved in the pathogenesis of pathological cardiac hypertrophy through regulating the production of reactive oxygen species (ROS) [10], suggesting it may participate in other cardiovascular diseases. We revealed that SIRT4 was able to ameliorate MI/R injury in vivo, expanding its role in another cardiovascular disease. SIRT4 is a mitochondrial-localized sirtuin and is functionally versatile [19]. We next found that SIRT4 preserved mitochondrial function and reduced cardiomyocyte apoptosis, therefore providing two possible mechanisms which may account for its protective function against MI/R injury. The preservation of mitochondrial function is an important aspect in myocardial cytoprotection [20]. The mitochondria are central sites for not only cellular energy production but also for controlling cell death [21]. Maintaining the homeostasis of oxidative phosphorylation within mitochondria to prevent cardiomyocyte death during ischemic injury has long been viewed as a critical event after myocardial infarction [22]. We discovered that mitochondria overexpressing SIRT4 and isolated from mice subjected to MI/R displayed preserved mitochondrial function as measured by increased complex I and II efficiency, and vice versa,
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
6
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Fig. 4. Sirt4 reduces cardiomyocyte apoptosis after MI/R. (A-F) Mice hearts were transfected in vivo with Ad-vector or Ad-SIRT4 (AeC), or with siCtrl or siSIRT4 (DeE). 3 days later, mice were subjected to sham surgery or experimental MI-R. Each group contained 5 mice. After 2 days, mice were analyzed as follows. (A and D) Representative images of TUNEL-stained heart sections from 4 groups. Apoptotic nuclei were identified by TUNEL staining (green) and total nuclei by DAPI counterstaining (blue). (B and E) Quantitative analysis of apoptotic nuclei. TUNEL-positive nuclei are expressed as a percentage of the total number of nuclei. Data were compared with Ad-vector group. Data are mean ± s.d. n ¼ 5. **, P < 0.01; NS, not significant. (C and F) Cardiomyocytes were isolated and analyzed by Western blotting to detect levels of SIRT4, Cleaved caspase3 and Caspase3. b-actin was used as a loading control. The representative images (left) and the statistical analysis of relative intensity of Cleaved caspase3/Caspase3 (right) were shown. Data were compared with Ad-vector group. Data are mean ± s.d. n ¼ 5. **, P < 0.01; NS, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
SIRT4 deficiency exhausted mitochondrial function. These lines of evidence suggest a role of preserving the mitochondrial function in the observed cytoprotection. Detection of mitochondrial structure in this scenario may provide useful evidence to further demonstrate whether SIRT4 preserves mitochondrial function after MI/R. We speculate that one possible mechanism for SIRT4 protective role in mitochondrial function may lie in its ability to modulate cellular respiration during reperfusion. It has been shown that the suppressed mitochondrial respiration protects against MI-R injury by limiting ROS production and mitochondrial uncoupling degree, which lead to decreased infarct size and preserved mitochondrial function [23]. However, to what extent the ROS production [10] and mitochondrial uncoupling [24] regulated by SIRT4 contribute to its role in MI/R need further interrogations. The protective role of SIRT4 was further reinforced by the decreased cleaved caspase3 and a decrease number of apoptotic cardiomyocytes. These suggest that SIRT4 is capable of inhibiting the progression of apoptosis after MI-R injury, at any rate, which we believe is associated with its function in preserving mitochondrial function. Nevertheless, direct evidence is required to better understand their connections. More importantly, deconvoluting the context-dependent roles of SIRT4 related to mitochondrial function and cell apoptosis may shed light on its mechanism of action in MIR. In conclusion, our study uncovers the protective role of SIRT4 in MI-R, and relates this effect to mitochondrial protection and reduced apoptosis. Thus, we propose here that enhancing the expression of SIRT4 in cardiomyocytes might be beneficial for reducing cardiomyocyte apoptosis and alleviating MI-R injury in clinic practice.
Disclosure of conflict of interest None. Acknowledgments This research was supported by the fund of Shaanxi science and technology support project (2017JCYJ007). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.113. References [1] M. Nichols, N. Townsend, P. Scarborough, M. Rayner, Cardiovascular disease in Europe: epidemiological update, Eur. Heart J. 34 (2013) 3028e3034. [2] D.J. Hausenloy, D.M. Yellon, Myocardial ischemia-reperfusion injury: a neglected therapeutic target, J. Clin. Invest. 123 (2013) 92e100. [3] R.A. Gottlieb, Cell death pathways in acute ischemia/reperfusion injury, J. Cardiovasc. Pharmacol. Therapeut. 16 (2011) 233e238. [4] E.J. Lesnefsky, Q. Chen, B. Tandler, C.L. Hoppel, Mitochondrial dysfunction and myocardial ischemia-reperfusion: implications for novel therapies, Annu. Rev. Pharmacol. Toxicol. 57 (2017) 535e565. [5] V. Borutaite, A. Jekabsone, R. Morkuniene, G.C. Brown, Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia, J. Mol. Cell. Cardiol. 35 (2003) 357e366. [6] D.A. Kubli, A.B. Gustafsson, Mitochondria and mitophagy: the yin and yang of cell death control, Circ. Res. 111 (2012) 1208e1221. [7] A. Cattelan, G. Ceolotto, S. Bova, M. Albiero, M. Kuppusamy, S. De Martin, A. Semplicini, G.P. Fadini, S.V. de Kreutzenberg, A. Avogaro, NAD(þ)-dependent SIRT1 deactivation has a key role on ischemia-reperfusion-induced apoptosis, Vasc. Pharmacol. 70 (2015) 35e44.
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113
G. Zeng et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7 [8] M.C. Haigis, D.A. Sinclair, Mammalian sirtuins: biological insights and disease relevance, Annu. Rev. Pathol. 5 (2010) 253e295. [9] A. Csibi, S.M. Fendt, C. Li, G. Poulogiannis, A.Y. Choo, D.J. Chapski, S.M. Jeong, J.M. Dempsey, A. Parkhitko, T. Morrison, E.P. Henske, M.C. Haigis, L.C. Cantley, G. Stephanopoulos, J. Yu, J. Blenis, The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4, Cell 153 (2013) 840e854. [10] Y.X. Luo, X. Tang, X.Z. An, X.M. Xie, X.F. Chen, X. Zhao, D.L. Hao, H.Z. Chen, D.P. Liu, SIRT4 accelerates Ang II-induced pathological cardiac hypertrophy by inhibiting manganese superoxide dismutase activity, Eur. Heart J. 38 (2017) 1389e1398. [11] J.W. Elrod, J.W. Calvert, J. Morrison, J.E. Doeller, D.W. Kraus, L. Tao, X. Jiao, R. Scalia, L. Kiss, C. Szabo, H. Kimura, C.W. Chow, D.J. Lefer, Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 15560e15565. [12] R. Shibata, K. Sato, D.R. Pimentel, Y. Takemura, S. Kihara, K. Ohashi, T. Funahashi, N. Ouchi, K. Walsh, Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms, Nat. Med. 11 (2005) 1096e1103. [13] S. Gao, X. Qi, J. Li, L. Sang, Upregulated KAT7 in synovial fibroblasts promotes Th17 cell differentiation and infiltration in rheumatoid arthritis, Biochem. Biophys. Res. Commun. 489 (2017) 235e241. [14] M.F. Corsten, R. Dennert, S. Jochems, T. Kuznetsova, Y. Devaux, L. Hofstra, D.R. Wagner, J.A. Staessen, S. Heymans, B. Schroen, Circulating MicroRNA208b and MicroRNA-499 reflect myocardial damage in cardiovascular disease, Circ. Cardiovasc. Genet. 3 (2010) 499e506.
7
[15] A. Kasahara, L. Scorrano, Mitochondria: from cell death executioners to regulators of cell differentiation, Trends Cell Biol. 24 (2014) 761e770. [16] R.H. Houtkooper, E. Pirinen, J. Auwerx, Sirtuins as regulators of metabolism and healthspan, Nature reviews, Mol. Cell Biol. 13 (2012) 225e238. [17] S. Nemoto, M.M. Fergusson, T. Finkel, Nutrient availability regulates SIRT1 through a forkhead-dependent pathway, Science 306 (2004) 2105e2108. [18] L.M. Buja, Myocardial ischemia and reperfusion injury, Cardiovasc. Pathol. : The Official Journal of the Society for Cardiovascular Pathology 14 (2005) 170e175. [19] S. Kumar, D.B. Lombard, For certain, SIRT4 activities!, Trends Biochem. Sci. 42 (2017) 499e501. [20] E. Murphy, C. Steenbergen, Preconditioning: the mitochondrial connection, Annu. Rev. Physiol. 69 (2007) 51e67. [21] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99e163. [22] R.B. Jennings, C.E. Ganote, Structural changes in myocardium during acute ischemia, Circ. Res. 35 (Suppl 3) (1974) 156e172. [23] Q. Chen, A.K. Camara, D.F. Stowe, C.L. Hoppel, E.J. Lesnefsky, Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion, American journal of physiology, Cell Physiol. 292 (2007) C137eC147. [24] A. Lang, R. Anand, S. Altinoluk-Hambuchen, H. Ezzahoini, A. Stefanski, A. Iram, L. Bergmann, J. Urbach, P. Bohler, J. Hansel, M. Franke, K. Stuhler, J. Krutmann, J. Scheller, B. Stork, A.S. Reichert, R.P. Piekorz, SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy, Aging 9 (2017) 2163e2189.
Please cite this article in press as: G. Zeng, et al., Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.113