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Inhibition of Drp1 attenuates mitochondrial damage and myocardial injury in Coxsackievirus B3 induced myocarditis
Biochemical and Biophysical Research Communications 484 (2017) 550e556
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of Drp1 attenuates mitochondrial damage and myocardial injury in Coxsackievirus B3 induced myocarditis Lin Lin, Ming Zhang, Rui Yan, Hu Shan, Jiayu Diao, Jin Wei* Department of Cardiology, The Second Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710004, China
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Article history: Received 15 January 2017 Accepted 22 January 2017 Available online 25 January 2017
Viral myocarditis (VMC) is closely related to apoptosis, oxidative stress, innate immunity, and energy metabolism, which are all linked to mitochondrial dysfunction. A close nexus between mitochondrial dynamics and cardiovascular disease with mitochondrial dysfunction has been deeply researched, but there is still no relevant report in viral myocarditis. In this study, we aimed to explore the role of Dynamin-related protein 1 (Drp1)-linked mitochondrial fission in VMC. Mice were inoculated with the Coxsackievirus B3 (CVB3) and treated with mdivi1 (a Drp1 inhibitor). Protein expression of Drp1 was increased in mitochondria while decreased in cytoplasm and accompanied by excessive mitochondrial fission in VMC mice. In addition, midivi1 treatment attenuate inflammatory cells infiltration in myocardium of the mice, serum Cardiac troponin I (CTnI) and Creatine kinase-MB (CK-MB) level. Mdivi1 also could improved the survival rate of mice and mitochondrial dysfunction reflected as the upregulated mitochondrial marker enzymatic activities of succinate dehydrogenase (SDH), cytochrome c oxidase (COX) and mitochondrial membrane potential (MMP). At the same time, mdivi1 rescued the body weight loss, myocardial injury and apoptosis of cardiomyocyte. Furthermore, decease in LVEDs and increase in EF and FS were detected by echocardiogram, which indicated the improved myocardial function. Thus, Drp1-linked excessive mitochondrial fission contributed to VMC and midivi1 may be a potential therapeutic approach. © 2017 Elsevier Inc. All rights reserved.
Keywords: Viral myocarditis Drp1 Mdivi1 Mitochondrial damage
1. Introduction Viral myocarditis (VMC), a worldwide cause of cardiac disease refers to pathogen induced heart-specific inflammatory condition, is considered to be a principal cause of heart failure in infants, children and young adults [1]. CVB3 infection is the most commonly identified pathogen, and the same virus strain was used to induce inflammatory heart disease in BALB/c mice with a spectrum of clinical manifestations remarkably similar to human [2]. Virus-induced cardiomyocyte damage and followed immune response plays an important role in the pathogenesis and progression of VMC [3]. However, intracellular processes and the mechanisms induced by CVB3 in heart tissue injury and organ dysfunction are still poorly understood. It is even worse, there is no effective therapy for these diseases to date.
* Corresponding author. Department of Cardiology, The Second Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710004, China. Tel: þ86 029-87679770 E-mail address: [email protected] (J. Wei). http://dx.doi.org/10.1016/j.bbrc.2017.01.116 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Mitochondria is central to multiple pathophysiological procedures in cellular metabolism, aging, innate immunity, energy production, cell apoptosis and other signaling pathways [4]. New focus of researches on mitochondria have discovered that mitochondria are highly dynamic organelles undergoing constant fission and fusion events, which is sensitive to the changes in the cellular physiological or metabolic conditions. Accordingly, the mitochondrial function and quality are preserved by this subtle balance between these two opposing processes due to this structure-function pattern. The central players of mitochondria fission include the Drp1 and mitochondrial fission 1 protein (Fis1). The vital players commandeering fusion include optic atrophy 1 protein (OPA1) and mitofusins (Mfn1 and Mfn2). As the key participant of mitochondrial fission, Drp1 is located in the cytoplasm (where it is primarily located) and recruited to mitochondria which is mediated by conformational changes linked to the GTPase cycle. In this manner, Drp1 is self-assembled and recruited to mitochondria resulting in an Drp1-dependent mitochondrial division and mitochondrial fragmentation [5]. In the past decades, the mitochondrial dynamics has been
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broadly studied in multifarious pathological states. Excessive mitochondrial fission is involved in ischemia/reperfusion injury [6], diabetic cardiomyopathy [7], pulmonary hypertension [8], heart failure [9], acute spinal cord injury [10], and neurodegenerative disorders [11]. Meanwhile, mdivi1, a small molecule identified and characterized as the selectively inhibitor of Drp1, has presented prominent curative effect in these wide array of disease models. Moreover, pathological damages caused by viral infections are in tandem with mitochondrial dynamic that have been described in few viral infections [12]. Our previous study has demonstrated that impairment of myocardial mitochondria is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction [13]. Nevertheless, since there is no report in whether CVB3 infection could disrupt the homeostasis of mitochondrial dynamic and promote Drp1 activation up to now, in addition, almost no biological function of mdivi1 has been disclosed in myocardial injury and organ dysfunction related to CVB3 infection, it is of great interest to explore the possible therapeutic function of mdivi1 on CVB3-induced acute viral myocarditis. In this study, the role of Drp1-linked mitochondrial fission was examined by acute viral infection of CVB3 in BALB/c mice. Our results suggests that perturbation of mitochondrial dynamic is involved in the pathological process of viral myocarditis. While inhibition of Drp1 by mdivi1 may have protective effect against CVB3-induced myocardial dysfunction partly. The underlying molecular mechanism may be associated with alleviation of mitochondrial damage, myocardial injury, and suppression of cardiomyocyte apoptosis. 2. Materials and methods 2.1. Mice and virus CVB3 (Nancy strain), which were generously provided by the Key Laboratory of Viral Heart Diseases, Zhongshan Hospital, was propagated in HeLa cells (ATCC number: CCL-2). Viral titers were routinely determined by a 50% tissue culture infectious dose (TCID50) assay. Specific pathogen free (SPF) male BALB/c mice (6e8 weeks, 18e23 g) were purchased and fed at the experimental animal center (Shanghai Medical College of Fudan University). Mdivi1 was purchased from Enzo life sciences (Branch office in Beijing, Shanghai, China). 2.2. Animal studies All experiments carried out in this study were strictly performed in a manner to minimize suffering of laboratory mice. All animal procedures were performed according to the Guide for the Care and Use of Medical Laboratory Animals and with the ethical approval of the Shanghai Medical Laboratory Animal Care and Use Committee. Firstly, one hundred mice were housed in a 22 C constant temperature SPF animal room with free access to distilled water and food for 3 days to accommodate to the circumstance of the animal lab. Then, all animals were randomly assigned to four groups: control group (n ¼ 10), VMC group (n ¼ 40), VMC þ mdivi1 (n ¼ 40), Mdivi1 (n ¼ 10). Concretely, mice in VMC group were intraperitoneally injected with 0.2 mL of eagle solution containing CVB3 (102 TCID50); After CVB3 infection, mice of VMC þ mdivi1 and mdivi1 group were injected with mdivi1 at the dose of 50 mg/kg/day for 14 d. The mdivi1 was dissolved in di-methyl sulfoxide (DMSO) which diluted with saline to ensure the final concentration of DMSO was less than 0.01%. Mice in control group were injected with the equal volume of eagle medium without CVB3 and DMSO. Dosage of mdivi1 were determined following previous studies [9].
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2.3. Echocardiogram A VEVO 770 ultrasound machine (Visual Sonic, Canada) was used to perform the echocardiographic study by an investigator blinded to the treatments. Mice were anaesthetized with isofluran (including 2%, maintenance 0.5%e1%) and the chest was shaved with depilatory cream. The parasternal long-axis view were conducted by two-dimensional, targeted M-mode tracings with a 30 MHz transducer (RMV 707B). The left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameters (LVEDd), left ventricular ejection fraction (EF) and fractional shortening (FS) were detected for 5e10 consecutive cardiac cycles. Each measurement data were averaged from three consecutive heart beats. 2.4. CTnI and CK-MB measurement The levels of cardiac injury biomarkers such as CTnI and CK-MB were detected by using commercially available kits (Nanjing Jiancheng Biology Engineer Institute, China). 2.5. Myocardial histopathology and TUNEL assay Fourteen days after CVB3 infection, the heart tissues were collected from mice and fixed in 4% paraformaldehyde, routinely processed and stained with hematoxylin and eosin (HE). Images were acquired by a light microscope equipment with a digital video camera (Olympus, Tokyo, Japan). Apoptosis of cardiomyocyte in heart tissue was determined by using a commercial fluorometric TUNEL system kit (Roche, Switzerland) according to the manufacturer's instructions. The apoptotic index was calculated by the positive cells rate under fluorescence microscopy. All the sections were examined by two independent investigators in a blinded manner. 2.6. Electron microscopy Myocardium from heart apex were harvested for ultrastructural analyses to quantify mitochondrial fission. Tissues were cut into 1 mm3 pieces and fixed in 2.5% glutaraldehyde, followed by 1% perosmic acid and a series of ethanol. 50 nm ultrathin slices were prepared and stained with uranyl acetate and lead citrate as we reported previously [14]. Five sections from each heart were selected and prepared for further heart ultrastructural analyses. We examined samples at a magnification of 12,000 with transmission electron microscope (HITACHI-H7650, Tokyo, Japan). Excessive mitochondrial fission was determined as the percentage of smaller than 0.6 mm2 in every 100 mitochondria measuring by a computer imaging analysis system. 2.7. Real-time PCR The total RNA was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcription was carried out using an RTPCR kit (TaKaRa, Japan). Real-time PCR was performed with SYBRExScript™ RT-PCR Kit (TaKaRa, Japan) on an iQ5 Multicolor RealTime PCR Detection System (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. The comparative 2DDct method was utilized to analyze the relative expression of genes. 2.8. Western blot analysis The total mitochondrial protein and cytoplastic protein were extracted using commercial Tissue/cell Mitochondria Isolation Kit (GENMED, Shanghai, China), and then separated on polyacrylamide
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L. Lin et al. / Biochemical and Biophysical Research Communications 484 (2017) 550e556
gels and transferred to a PVDF membrane for detection with various antibodies, including primary antibodies for Drp1 (1:1000, Cell Signaling Technology corporation, USA), b-actin, VDAC (1:1000, Pioneer Biotechnology, Xi'an, China). The PVDF membrane was gently incubated overnight at 4 C with primary antibody and incubated with HRP-conjugated anti-rabbit IgG antibody (Zhuangzhi Biotechnology, Xi'an, China). The values of band intensities were detected by enhanced chemiluminescence (ECL) and quantized by quantity one. 2.9. Mitochondrial membrane potential and enzyme activities detection Mitochondria was obtained from freshly prepared heart tissue. Briefly, after separation of nuclei and unbroken cells debris by centrifugation at 1000g for 10 min at 4 C, the mitochondrial fraction was acquire by centrifugation of supernatant at 10000g for 10 min at 4 C, suspended in mitochondrial storage fluid (300 mmol/L sucrose, 2 mmol/L Hepes, 0.1 mmol/L EGTA, pH7.4). Then, the activities of COX and SDH were measured respectively by using a COX assay kit (GENEMED Scientifics Inc., Shanghai, China) and SDH assay kit (Jiancheng Bioengineering Co., Nanjing. China). The MMP was identified with the lipophilic cationic probe JC-1 as described in our previous papers [14]. 2.10. Statistical analysis Data were shown as the mean ± standard deviation (SD). Statistical analysis of the data were performed using the GraphPad Prism 5.0 statistical program. Two groups were compared using the Student's t-test. Multiple groups were performed by one-way analysis of variance (ANOVA), followed by an LSD test. The
survival rates of CVB3 infected mice were compared and analyzed with Kaplan-Meier plot. A value was considered significant with P < 0.05. 3. Result 3.1. Excessive mitochondrial fission in myocardium of VMC mice In our study, mice were administered intraperitoneal injection with CVB3 to establish the VMC animal model. Then, the heart tissues were obtained quickly. To examine whether disturbed mitochondrial dynamics profile is involved in myocardium of CVB3-infected group mice, firstly, we examined the mitochondrial network by electron microscopy. Our results showed the obvious sarcomere fragmentation and inhomogeneous myofibril dissolved. Meanwhile, the structure of mitochondria have significantly changed with vesicle, pyknosis, and the decreased matrix in myocardium in VMC mice (Fig. 1A). In addition, compared with the control group, the size of the mitochondria decreased significantly and the percentage of fragmented mitochondria increased by 26% (Fig. 1C). Next, real-time PCR was used for detecting the genes mRNA expression. We found a 3.2-fold increase of Drp1, a 1.5-fold increase of Fis1 and a significant 0.7-fold decrease of Mfn1 in VMC group. Whereas Mfn2 had no difference between VMC group and control group (Fig. 1B). All these findings indicated that excessive mitochondrial fission was involved in myocardium of VMC mice. 3.2. Translocation of Drp1 from cytoplasm to mitochondria and excessive mitochondrial fission in VMC It has been previously found that the cytoplasmic Drp1
Fig. 1. Excessive mitochondrial fission involved in heart tissue of CVB3-infected mice on day 14. A. The morphology and structure of mitochondria in myocardium of VMC and control group were detected by transmission electron microscopy (magnification: 12,000 ). B. The related mRNA expression of important molecules commandeering fusion and fission (Fis1, Drp1, Mfn1, Mfn2) was detected by Real-time PCR. C. The percentage of mitochondria smaller than 0.6 mm2 from 100 mitochondria in a representative area. D. Expression of protein level of Drp1 in mitochondria fraction and cytoplasm of heart tissue were detected by Western blot analysis. b-actin and VDAC were used as internal control of cytoplasm and mitochondrial fraction respectively. Results were presented as the mean ± SD. *P < 0.05 and **P < 0.01 vs. control group.
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Fig. 2. Mdivi1 treatment mediated protection against CVB3-induced myocarditis. A. Paraffin sections of heart tissues revealed cardiac inflammation by H&E staining (magnification: 100 ). B. The body weight changes were measured every two days until 14 d after CVB3 infection. C. The serum CTnI was detected on day 14 after CVB3 infection. D. The survival rate was measured every day until d 14, and no more mice died after day 8. E. The CK-MB was detected on day 14 after CVB3 infection. Results were presented as the mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; #p < 0.05 vs. VMC group.
accumulates in mitochondrial outer membrane and consequently mediates activation of the mitochondrial fission process [15]. To further explore the mechanism of Drp1 translocation in VMC, we examined Drp1 protein expression in cytoplasmic fraction and freshly isolated mitochondrial fraction of heart tissue respectively. Western blot indicated that Drp1 was increased significantly in mitochondria while decreased in cytoplasm in VMC group (Fig. 1D). These results suggested that CVB3 infection prompted Drp1 translocation from cytoplasm to mitochondrial to stimulated Drp1linked mitochondrial fission. 3.3. Effect of mdivi1on inflammatory cell infiltration and myocardium injury of VMC mice To investigate the pathological significances of excessive mitochondria, mdivi1was administrated to VMC mice. On day 14, we found that mdivi1 treatment could obviously mitigate infiltration of inflammatory cell in myocardium compared with VMC group (Fig. 2A). Furthermore, mdivi1 treatment significantly decreased the serum cTnI (Fig. 2C) and CK-MB levels (Fig. 2E). The VMC mice lost the body weight dramatically, and even reached to 28.2%, while the mdivi1 rescued the lost weight of VMC mice (Fig. 2B). At the same time, more than 70% mice died within 8 days in VMC group, as expected, mdivi1 treatment could significantly improve survival rate (Fig. 2D). These results implied that mdivi1 treatment could effectively protect mice from lethal myocarditis after CVB3 infection. 3.4. Effect of mdivi1 treatment on cardiomyocyte apoptosis and mitochondrial damage in VMC mice
Fig. 3. Effects of mdivi1 treatment on apoptosis in CVB3 induced myocarditis. A. Representative photomicrographs (magnification: 200 ) of TUNEL staining in myocardial tissue of each group. TUNEL-positive apoptotic nuclei were stained with green fluorescence, the nuclei were stained with blue fluorescence. B. Apoptotic index measured from 100 nucleus in a representative area. Results were presented as the mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; #p < 0.05 vs. VMC group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fluorescence TUNEL assay showed that, in comparison with control group and mdivi1 group, the apoptotic cells increased significantly in VMC group than in control group (Fig. 3A). Mdivi1 treatment inhibited percentage of cardiomyocyte apoptosis induced by CVB3 infection (Fig. 3B). We further investigated the effect of mdivi1 treatment on mitochondrial function. MMP, enzymatic activities of the COX and SDH were measured in each group. Compared with the control group, both COX and SDH activities
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Fig. 4. Effects of mdivi1 treatment on mitochondrial damage and cardiac function of VMC mice. A. Representative M-mode images and changes of echocardiographic parameters in each group. B. The LVEDs, C. the EF and FS were detected by echocardiography. D and E. The enzymatic activities of COX and SDH in isolated cardiac mitochondria from mice. F. The MMP in cardiac mitochondria among each group. Data are expressed as mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; #p < 0.05 vs. VMC group.
were remarkably decreased in VMC mice (Fig. 4D and E). Moreover, MMP was obviously decreased in VMC mice (Fig. 4F). Accordingly, enzymatic activities of COX and SDH, as well as MMP were downregulated by mdivi1 treatment. 3.5. Mdivi1 protects against CVB3 induced myocardial dysfunction Echocardiogram were performed to examine the effect of mdivi1 on myocardial function. The data showed that VMC mice presented a significant left ventricular systolic dysfunction. Specifically, LVEDs in VMC mice increased markedly than in control group (Fig. 4B); EF and FS, the most important parameters of myocardial function, were significantly decreased in CVB3 infected mice (Fig. 4C). All these alterations, including LVEDs, EF and FS caused by CVB3 infection could be reversed partly by mdivi1 treatment. These our results indicated that mdivi1 could partly reverse CVB3-mediated myocardial dysfunction. Collectively, our findings suggested that Drp1-linked mitochondrial fission is involved in VMC and mdivi1 treatment could attenuate myocardial injury and preserved myocardial function. 4. Discussion The pathogenesis of VMC is multi-factorial and not fully understood by now. For the past few years, researches about excessive mitochondrial fission were widely detected in many cardiovascular diseases, including in ischemia/reperfusion injury [16], myocardial infarction [17], and heart failure [18]. However, it is unclear about
the influence of CVB3 infection on mitochondrial dynamics in VMC. Given that Drp1-linked mitochondrial fission is a common stress response to various pathological states and closely related with cellular homeostasis, mitochondrial function, innate immunity, apoptosis [19,20]. In the present study, we found that excessive mitochondrial fission was involved in VMC. Moreover, mdivi1 treatment significantly inhibited the Drp1-linked mitochondrial damage, inflammation, cardiomyocyte apoptosis and rescued myocardial dysfunction in VMC mice. Studies have shown that a range of virus infections perturb the mitochondrial dynamics and distort mitochondrial functions to facilitate its own replication and survival in host cell [21]. For example, previous studies shown that both hepatitis C virus and hepatitis B virus could promote mitochondrial fission by inducing Drp-1 S616 phosphorylation and further contribute to maintenance of persistent viral infection [22,23]. In addition, latent membrane protein 2A (LMP2A) of EpsteineBarr virus (EBV) induced Drp1 expression and mitochondrial fission which contributed to EBVassociated gastric and breast carcinomas [24]. In accordance with the previous researches, we observed the Drp1-linked mitochondrial fission in VMC for the first time. These observations collectively implicate perturbation of mitochondrial dynamics may provide a survival strategy in the virus-host battle after CVB3 infection. Evidences suggest that mitochondria appear to act as a platform for innate immunity response for antiviral defense. It has been identified that mitochondrial dynamics regulate antiviral signaling [25]. It is known that MMP is a basic function of the mitochondria.
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In RNA viral infection, MMP is required for MAVS (mitochondrial antiviral signaling)-mediated antiviral signaling to trigger the production of type I interferons [26]. Interestingly, Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP [3]. Inhibition of Drp1 with mdivi1 may affect MMP dissipation and further regulate innate immunity response of VMC after CVB3 infection. A previous research of our team reported that mitochondrial damage is an important pathological mechanism leading to myocardial injury and cardiac dysfunction associated with mitochondrial DNA (mtDNA) deletion and the loss of mitochondrial membrane phospholipids in VMC [13]. In addition, the catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex IV, while SDH, essential for assembly of the complex II, increase might indicate mitochondrial biogenesis [27]. Consist with these reports, alterations in energy production arise already in the acute phase of viral myocarditis [28]. It implied changes of activities of COX and SDH may contribute the impairment of mitochondrial energy production. Another research has revealed that all myocardial respiratory chain complex (RC) activities were restricted in CVB3infected A.SW/SnJ mice. The study revealed that decreased RC activities were correlated with increased CVB3 titer which are remarkably linked to myocardial inflammation and myocardial dysfunction even determined the fate of the VMC progression. In this study, mdivi1 treatment could partly rescue mitochondrial enzymatic activities of SDH and COX, MMP, inflammatory cells infiltration in myocardium, as well as serum CTnI and CK-MB in VMC mice, which is in agreement with the previous findings and logical that midivi1 treatment attenuated the myocardial injury due to mitigate the excessive mitochondrial fission. Our present study also found that mdivi1 treatment significantly decreased the cardiomyocyte apoptosis in VMC mice. In fact, it is common that the cytopathic injury in early phase of CVB3induced VMC mainly attribute to apoptosis and viral replication [29]. A previous research has shown that inhibition of mitochondrial fission mediated by Drp1 can limit reactive oxygen species (ROS) production and apoptosis in cardiomyocyte [30]. In addition, higher rates of cardiomyocyte apoptosis are associated with the development of fatal heart failure in acute human myocarditis [31]. During apoptosis, a pro-apoptotic Bcl-2 family member, Bax colocalizes with Drp1 at fission sites responsible for release of Cytochrome c [32], which is an important early event in Caspase 3 activation, finally induce apoptosis, contributing to the loss of hostcell viability and progeny virus release in VMC [33]. In conclusion, our present study reveals that Drp1-linked excessive mitochondrial fission contributes to VMC in mice. Mdivi1 treatment protects against myocardial injury and preserves myocardial function in VMC may associated with inhibiting mitochondrial damage, inflammation, apoptosis and innate immunity. Since concrete mechanisms in mdivi1 treatment are not yet realized, further and deeper studies are required to elucidate the mitochondria and virus-host interaction. This novel finding will not only help us better understand the pathogenesis of VMC but also shed a new light on possible therapeutic strategies in VMC. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was supported by the Special Fund of Talentdevelopment of the second affiliated hospital of Xi'an Jiaotong University (No.6YJ(ZD), and grants from National Natural Science Foundation of China (No. 81170209 to J.W. and No. 81100210 to J.X.).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of Drp1 attenuates mitochondrial damage and myocardial injury in Coxsackievirus B3 induced myocarditis Lin Lin, Ming Zhang, Rui Yan, Hu Shan, Jiayu Diao, Jin Wei* Department of Cardiology, The Second Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710004, China
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a b s t r a c t
Article history: Received 15 January 2017 Accepted 22 January 2017 Available online 25 January 2017
Viral myocarditis (VMC) is closely related to apoptosis, oxidative stress, innate immunity, and energy metabolism, which are all linked to mitochondrial dysfunction. A close nexus between mitochondrial dynamics and cardiovascular disease with mitochondrial dysfunction has been deeply researched, but there is still no relevant report in viral myocarditis. In this study, we aimed to explore the role of Dynamin-related protein 1 (Drp1)-linked mitochondrial fission in VMC. Mice were inoculated with the Coxsackievirus B3 (CVB3) and treated with mdivi1 (a Drp1 inhibitor). Protein expression of Drp1 was increased in mitochondria while decreased in cytoplasm and accompanied by excessive mitochondrial fission in VMC mice. In addition, midivi1 treatment attenuate inflammatory cells infiltration in myocardium of the mice, serum Cardiac troponin I (CTnI) and Creatine kinase-MB (CK-MB) level. Mdivi1 also could improved the survival rate of mice and mitochondrial dysfunction reflected as the upregulated mitochondrial marker enzymatic activities of succinate dehydrogenase (SDH), cytochrome c oxidase (COX) and mitochondrial membrane potential (MMP). At the same time, mdivi1 rescued the body weight loss, myocardial injury and apoptosis of cardiomyocyte. Furthermore, decease in LVEDs and increase in EF and FS were detected by echocardiogram, which indicated the improved myocardial function. Thus, Drp1-linked excessive mitochondrial fission contributed to VMC and midivi1 may be a potential therapeutic approach. © 2017 Elsevier Inc. All rights reserved.
Keywords: Viral myocarditis Drp1 Mdivi1 Mitochondrial damage
1. Introduction Viral myocarditis (VMC), a worldwide cause of cardiac disease refers to pathogen induced heart-specific inflammatory condition, is considered to be a principal cause of heart failure in infants, children and young adults [1]. CVB3 infection is the most commonly identified pathogen, and the same virus strain was used to induce inflammatory heart disease in BALB/c mice with a spectrum of clinical manifestations remarkably similar to human [2]. Virus-induced cardiomyocyte damage and followed immune response plays an important role in the pathogenesis and progression of VMC [3]. However, intracellular processes and the mechanisms induced by CVB3 in heart tissue injury and organ dysfunction are still poorly understood. It is even worse, there is no effective therapy for these diseases to date.
* Corresponding author. Department of Cardiology, The Second Affiliated Hospital, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710004, China. Tel: þ86 029-87679770 E-mail address: [email protected] (J. Wei). http://dx.doi.org/10.1016/j.bbrc.2017.01.116 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Mitochondria is central to multiple pathophysiological procedures in cellular metabolism, aging, innate immunity, energy production, cell apoptosis and other signaling pathways [4]. New focus of researches on mitochondria have discovered that mitochondria are highly dynamic organelles undergoing constant fission and fusion events, which is sensitive to the changes in the cellular physiological or metabolic conditions. Accordingly, the mitochondrial function and quality are preserved by this subtle balance between these two opposing processes due to this structure-function pattern. The central players of mitochondria fission include the Drp1 and mitochondrial fission 1 protein (Fis1). The vital players commandeering fusion include optic atrophy 1 protein (OPA1) and mitofusins (Mfn1 and Mfn2). As the key participant of mitochondrial fission, Drp1 is located in the cytoplasm (where it is primarily located) and recruited to mitochondria which is mediated by conformational changes linked to the GTPase cycle. In this manner, Drp1 is self-assembled and recruited to mitochondria resulting in an Drp1-dependent mitochondrial division and mitochondrial fragmentation [5]. In the past decades, the mitochondrial dynamics has been
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broadly studied in multifarious pathological states. Excessive mitochondrial fission is involved in ischemia/reperfusion injury [6], diabetic cardiomyopathy [7], pulmonary hypertension [8], heart failure [9], acute spinal cord injury [10], and neurodegenerative disorders [11]. Meanwhile, mdivi1, a small molecule identified and characterized as the selectively inhibitor of Drp1, has presented prominent curative effect in these wide array of disease models. Moreover, pathological damages caused by viral infections are in tandem with mitochondrial dynamic that have been described in few viral infections [12]. Our previous study has demonstrated that impairment of myocardial mitochondria is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction [13]. Nevertheless, since there is no report in whether CVB3 infection could disrupt the homeostasis of mitochondrial dynamic and promote Drp1 activation up to now, in addition, almost no biological function of mdivi1 has been disclosed in myocardial injury and organ dysfunction related to CVB3 infection, it is of great interest to explore the possible therapeutic function of mdivi1 on CVB3-induced acute viral myocarditis. In this study, the role of Drp1-linked mitochondrial fission was examined by acute viral infection of CVB3 in BALB/c mice. Our results suggests that perturbation of mitochondrial dynamic is involved in the pathological process of viral myocarditis. While inhibition of Drp1 by mdivi1 may have protective effect against CVB3-induced myocardial dysfunction partly. The underlying molecular mechanism may be associated with alleviation of mitochondrial damage, myocardial injury, and suppression of cardiomyocyte apoptosis. 2. Materials and methods 2.1. Mice and virus CVB3 (Nancy strain), which were generously provided by the Key Laboratory of Viral Heart Diseases, Zhongshan Hospital, was propagated in HeLa cells (ATCC number: CCL-2). Viral titers were routinely determined by a 50% tissue culture infectious dose (TCID50) assay. Specific pathogen free (SPF) male BALB/c mice (6e8 weeks, 18e23 g) were purchased and fed at the experimental animal center (Shanghai Medical College of Fudan University). Mdivi1 was purchased from Enzo life sciences (Branch office in Beijing, Shanghai, China). 2.2. Animal studies All experiments carried out in this study were strictly performed in a manner to minimize suffering of laboratory mice. All animal procedures were performed according to the Guide for the Care and Use of Medical Laboratory Animals and with the ethical approval of the Shanghai Medical Laboratory Animal Care and Use Committee. Firstly, one hundred mice were housed in a 22 C constant temperature SPF animal room with free access to distilled water and food for 3 days to accommodate to the circumstance of the animal lab. Then, all animals were randomly assigned to four groups: control group (n ¼ 10), VMC group (n ¼ 40), VMC þ mdivi1 (n ¼ 40), Mdivi1 (n ¼ 10). Concretely, mice in VMC group were intraperitoneally injected with 0.2 mL of eagle solution containing CVB3 (102 TCID50); After CVB3 infection, mice of VMC þ mdivi1 and mdivi1 group were injected with mdivi1 at the dose of 50 mg/kg/day for 14 d. The mdivi1 was dissolved in di-methyl sulfoxide (DMSO) which diluted with saline to ensure the final concentration of DMSO was less than 0.01%. Mice in control group were injected with the equal volume of eagle medium without CVB3 and DMSO. Dosage of mdivi1 were determined following previous studies [9].
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2.3. Echocardiogram A VEVO 770 ultrasound machine (Visual Sonic, Canada) was used to perform the echocardiographic study by an investigator blinded to the treatments. Mice were anaesthetized with isofluran (including 2%, maintenance 0.5%e1%) and the chest was shaved with depilatory cream. The parasternal long-axis view were conducted by two-dimensional, targeted M-mode tracings with a 30 MHz transducer (RMV 707B). The left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameters (LVEDd), left ventricular ejection fraction (EF) and fractional shortening (FS) were detected for 5e10 consecutive cardiac cycles. Each measurement data were averaged from three consecutive heart beats. 2.4. CTnI and CK-MB measurement The levels of cardiac injury biomarkers such as CTnI and CK-MB were detected by using commercially available kits (Nanjing Jiancheng Biology Engineer Institute, China). 2.5. Myocardial histopathology and TUNEL assay Fourteen days after CVB3 infection, the heart tissues were collected from mice and fixed in 4% paraformaldehyde, routinely processed and stained with hematoxylin and eosin (HE). Images were acquired by a light microscope equipment with a digital video camera (Olympus, Tokyo, Japan). Apoptosis of cardiomyocyte in heart tissue was determined by using a commercial fluorometric TUNEL system kit (Roche, Switzerland) according to the manufacturer's instructions. The apoptotic index was calculated by the positive cells rate under fluorescence microscopy. All the sections were examined by two independent investigators in a blinded manner. 2.6. Electron microscopy Myocardium from heart apex were harvested for ultrastructural analyses to quantify mitochondrial fission. Tissues were cut into 1 mm3 pieces and fixed in 2.5% glutaraldehyde, followed by 1% perosmic acid and a series of ethanol. 50 nm ultrathin slices were prepared and stained with uranyl acetate and lead citrate as we reported previously [14]. Five sections from each heart were selected and prepared for further heart ultrastructural analyses. We examined samples at a magnification of 12,000 with transmission electron microscope (HITACHI-H7650, Tokyo, Japan). Excessive mitochondrial fission was determined as the percentage of smaller than 0.6 mm2 in every 100 mitochondria measuring by a computer imaging analysis system. 2.7. Real-time PCR The total RNA was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcription was carried out using an RTPCR kit (TaKaRa, Japan). Real-time PCR was performed with SYBRExScript™ RT-PCR Kit (TaKaRa, Japan) on an iQ5 Multicolor RealTime PCR Detection System (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. The comparative 2DDct method was utilized to analyze the relative expression of genes. 2.8. Western blot analysis The total mitochondrial protein and cytoplastic protein were extracted using commercial Tissue/cell Mitochondria Isolation Kit (GENMED, Shanghai, China), and then separated on polyacrylamide
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gels and transferred to a PVDF membrane for detection with various antibodies, including primary antibodies for Drp1 (1:1000, Cell Signaling Technology corporation, USA), b-actin, VDAC (1:1000, Pioneer Biotechnology, Xi'an, China). The PVDF membrane was gently incubated overnight at 4 C with primary antibody and incubated with HRP-conjugated anti-rabbit IgG antibody (Zhuangzhi Biotechnology, Xi'an, China). The values of band intensities were detected by enhanced chemiluminescence (ECL) and quantized by quantity one. 2.9. Mitochondrial membrane potential and enzyme activities detection Mitochondria was obtained from freshly prepared heart tissue. Briefly, after separation of nuclei and unbroken cells debris by centrifugation at 1000g for 10 min at 4 C, the mitochondrial fraction was acquire by centrifugation of supernatant at 10000g for 10 min at 4 C, suspended in mitochondrial storage fluid (300 mmol/L sucrose, 2 mmol/L Hepes, 0.1 mmol/L EGTA, pH7.4). Then, the activities of COX and SDH were measured respectively by using a COX assay kit (GENEMED Scientifics Inc., Shanghai, China) and SDH assay kit (Jiancheng Bioengineering Co., Nanjing. China). The MMP was identified with the lipophilic cationic probe JC-1 as described in our previous papers [14]. 2.10. Statistical analysis Data were shown as the mean ± standard deviation (SD). Statistical analysis of the data were performed using the GraphPad Prism 5.0 statistical program. Two groups were compared using the Student's t-test. Multiple groups were performed by one-way analysis of variance (ANOVA), followed by an LSD test. The
survival rates of CVB3 infected mice were compared and analyzed with Kaplan-Meier plot. A value was considered significant with P < 0.05. 3. Result 3.1. Excessive mitochondrial fission in myocardium of VMC mice In our study, mice were administered intraperitoneal injection with CVB3 to establish the VMC animal model. Then, the heart tissues were obtained quickly. To examine whether disturbed mitochondrial dynamics profile is involved in myocardium of CVB3-infected group mice, firstly, we examined the mitochondrial network by electron microscopy. Our results showed the obvious sarcomere fragmentation and inhomogeneous myofibril dissolved. Meanwhile, the structure of mitochondria have significantly changed with vesicle, pyknosis, and the decreased matrix in myocardium in VMC mice (Fig. 1A). In addition, compared with the control group, the size of the mitochondria decreased significantly and the percentage of fragmented mitochondria increased by 26% (Fig. 1C). Next, real-time PCR was used for detecting the genes mRNA expression. We found a 3.2-fold increase of Drp1, a 1.5-fold increase of Fis1 and a significant 0.7-fold decrease of Mfn1 in VMC group. Whereas Mfn2 had no difference between VMC group and control group (Fig. 1B). All these findings indicated that excessive mitochondrial fission was involved in myocardium of VMC mice. 3.2. Translocation of Drp1 from cytoplasm to mitochondria and excessive mitochondrial fission in VMC It has been previously found that the cytoplasmic Drp1
Fig. 1. Excessive mitochondrial fission involved in heart tissue of CVB3-infected mice on day 14. A. The morphology and structure of mitochondria in myocardium of VMC and control group were detected by transmission electron microscopy (magnification: 12,000 ). B. The related mRNA expression of important molecules commandeering fusion and fission (Fis1, Drp1, Mfn1, Mfn2) was detected by Real-time PCR. C. The percentage of mitochondria smaller than 0.6 mm2 from 100 mitochondria in a representative area. D. Expression of protein level of Drp1 in mitochondria fraction and cytoplasm of heart tissue were detected by Western blot analysis. b-actin and VDAC were used as internal control of cytoplasm and mitochondrial fraction respectively. Results were presented as the mean ± SD. *P < 0.05 and **P < 0.01 vs. control group.
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Fig. 2. Mdivi1 treatment mediated protection against CVB3-induced myocarditis. A. Paraffin sections of heart tissues revealed cardiac inflammation by H&E staining (magnification: 100 ). B. The body weight changes were measured every two days until 14 d after CVB3 infection. C. The serum CTnI was detected on day 14 after CVB3 infection. D. The survival rate was measured every day until d 14, and no more mice died after day 8. E. The CK-MB was detected on day 14 after CVB3 infection. Results were presented as the mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; #p < 0.05 vs. VMC group.
accumulates in mitochondrial outer membrane and consequently mediates activation of the mitochondrial fission process [15]. To further explore the mechanism of Drp1 translocation in VMC, we examined Drp1 protein expression in cytoplasmic fraction and freshly isolated mitochondrial fraction of heart tissue respectively. Western blot indicated that Drp1 was increased significantly in mitochondria while decreased in cytoplasm in VMC group (Fig. 1D). These results suggested that CVB3 infection prompted Drp1 translocation from cytoplasm to mitochondrial to stimulated Drp1linked mitochondrial fission. 3.3. Effect of mdivi1on inflammatory cell infiltration and myocardium injury of VMC mice To investigate the pathological significances of excessive mitochondria, mdivi1was administrated to VMC mice. On day 14, we found that mdivi1 treatment could obviously mitigate infiltration of inflammatory cell in myocardium compared with VMC group (Fig. 2A). Furthermore, mdivi1 treatment significantly decreased the serum cTnI (Fig. 2C) and CK-MB levels (Fig. 2E). The VMC mice lost the body weight dramatically, and even reached to 28.2%, while the mdivi1 rescued the lost weight of VMC mice (Fig. 2B). At the same time, more than 70% mice died within 8 days in VMC group, as expected, mdivi1 treatment could significantly improve survival rate (Fig. 2D). These results implied that mdivi1 treatment could effectively protect mice from lethal myocarditis after CVB3 infection. 3.4. Effect of mdivi1 treatment on cardiomyocyte apoptosis and mitochondrial damage in VMC mice
Fig. 3. Effects of mdivi1 treatment on apoptosis in CVB3 induced myocarditis. A. Representative photomicrographs (magnification: 200 ) of TUNEL staining in myocardial tissue of each group. TUNEL-positive apoptotic nuclei were stained with green fluorescence, the nuclei were stained with blue fluorescence. B. Apoptotic index measured from 100 nucleus in a representative area. Results were presented as the mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; #p < 0.05 vs. VMC group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fluorescence TUNEL assay showed that, in comparison with control group and mdivi1 group, the apoptotic cells increased significantly in VMC group than in control group (Fig. 3A). Mdivi1 treatment inhibited percentage of cardiomyocyte apoptosis induced by CVB3 infection (Fig. 3B). We further investigated the effect of mdivi1 treatment on mitochondrial function. MMP, enzymatic activities of the COX and SDH were measured in each group. Compared with the control group, both COX and SDH activities
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Fig. 4. Effects of mdivi1 treatment on mitochondrial damage and cardiac function of VMC mice. A. Representative M-mode images and changes of echocardiographic parameters in each group. B. The LVEDs, C. the EF and FS were detected by echocardiography. D and E. The enzymatic activities of COX and SDH in isolated cardiac mitochondria from mice. F. The MMP in cardiac mitochondria among each group. Data are expressed as mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; #p < 0.05 vs. VMC group.
were remarkably decreased in VMC mice (Fig. 4D and E). Moreover, MMP was obviously decreased in VMC mice (Fig. 4F). Accordingly, enzymatic activities of COX and SDH, as well as MMP were downregulated by mdivi1 treatment. 3.5. Mdivi1 protects against CVB3 induced myocardial dysfunction Echocardiogram were performed to examine the effect of mdivi1 on myocardial function. The data showed that VMC mice presented a significant left ventricular systolic dysfunction. Specifically, LVEDs in VMC mice increased markedly than in control group (Fig. 4B); EF and FS, the most important parameters of myocardial function, were significantly decreased in CVB3 infected mice (Fig. 4C). All these alterations, including LVEDs, EF and FS caused by CVB3 infection could be reversed partly by mdivi1 treatment. These our results indicated that mdivi1 could partly reverse CVB3-mediated myocardial dysfunction. Collectively, our findings suggested that Drp1-linked mitochondrial fission is involved in VMC and mdivi1 treatment could attenuate myocardial injury and preserved myocardial function. 4. Discussion The pathogenesis of VMC is multi-factorial and not fully understood by now. For the past few years, researches about excessive mitochondrial fission were widely detected in many cardiovascular diseases, including in ischemia/reperfusion injury [16], myocardial infarction [17], and heart failure [18]. However, it is unclear about
the influence of CVB3 infection on mitochondrial dynamics in VMC. Given that Drp1-linked mitochondrial fission is a common stress response to various pathological states and closely related with cellular homeostasis, mitochondrial function, innate immunity, apoptosis [19,20]. In the present study, we found that excessive mitochondrial fission was involved in VMC. Moreover, mdivi1 treatment significantly inhibited the Drp1-linked mitochondrial damage, inflammation, cardiomyocyte apoptosis and rescued myocardial dysfunction in VMC mice. Studies have shown that a range of virus infections perturb the mitochondrial dynamics and distort mitochondrial functions to facilitate its own replication and survival in host cell [21]. For example, previous studies shown that both hepatitis C virus and hepatitis B virus could promote mitochondrial fission by inducing Drp-1 S616 phosphorylation and further contribute to maintenance of persistent viral infection [22,23]. In addition, latent membrane protein 2A (LMP2A) of EpsteineBarr virus (EBV) induced Drp1 expression and mitochondrial fission which contributed to EBVassociated gastric and breast carcinomas [24]. In accordance with the previous researches, we observed the Drp1-linked mitochondrial fission in VMC for the first time. These observations collectively implicate perturbation of mitochondrial dynamics may provide a survival strategy in the virus-host battle after CVB3 infection. Evidences suggest that mitochondria appear to act as a platform for innate immunity response for antiviral defense. It has been identified that mitochondrial dynamics regulate antiviral signaling [25]. It is known that MMP is a basic function of the mitochondria.
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In RNA viral infection, MMP is required for MAVS (mitochondrial antiviral signaling)-mediated antiviral signaling to trigger the production of type I interferons [26]. Interestingly, Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP [3]. Inhibition of Drp1 with mdivi1 may affect MMP dissipation and further regulate innate immunity response of VMC after CVB3 infection. A previous research of our team reported that mitochondrial damage is an important pathological mechanism leading to myocardial injury and cardiac dysfunction associated with mitochondrial DNA (mtDNA) deletion and the loss of mitochondrial membrane phospholipids in VMC [13]. In addition, the catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex IV, while SDH, essential for assembly of the complex II, increase might indicate mitochondrial biogenesis [27]. Consist with these reports, alterations in energy production arise already in the acute phase of viral myocarditis [28]. It implied changes of activities of COX and SDH may contribute the impairment of mitochondrial energy production. Another research has revealed that all myocardial respiratory chain complex (RC) activities were restricted in CVB3infected A.SW/SnJ mice. The study revealed that decreased RC activities were correlated with increased CVB3 titer which are remarkably linked to myocardial inflammation and myocardial dysfunction even determined the fate of the VMC progression. In this study, mdivi1 treatment could partly rescue mitochondrial enzymatic activities of SDH and COX, MMP, inflammatory cells infiltration in myocardium, as well as serum CTnI and CK-MB in VMC mice, which is in agreement with the previous findings and logical that midivi1 treatment attenuated the myocardial injury due to mitigate the excessive mitochondrial fission. Our present study also found that mdivi1 treatment significantly decreased the cardiomyocyte apoptosis in VMC mice. In fact, it is common that the cytopathic injury in early phase of CVB3induced VMC mainly attribute to apoptosis and viral replication [29]. A previous research has shown that inhibition of mitochondrial fission mediated by Drp1 can limit reactive oxygen species (ROS) production and apoptosis in cardiomyocyte [30]. In addition, higher rates of cardiomyocyte apoptosis are associated with the development of fatal heart failure in acute human myocarditis [31]. During apoptosis, a pro-apoptotic Bcl-2 family member, Bax colocalizes with Drp1 at fission sites responsible for release of Cytochrome c [32], which is an important early event in Caspase 3 activation, finally induce apoptosis, contributing to the loss of hostcell viability and progeny virus release in VMC [33]. In conclusion, our present study reveals that Drp1-linked excessive mitochondrial fission contributes to VMC in mice. Mdivi1 treatment protects against myocardial injury and preserves myocardial function in VMC may associated with inhibiting mitochondrial damage, inflammation, apoptosis and innate immunity. Since concrete mechanisms in mdivi1 treatment are not yet realized, further and deeper studies are required to elucidate the mitochondria and virus-host interaction. This novel finding will not only help us better understand the pathogenesis of VMC but also shed a new light on possible therapeutic strategies in VMC. Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was supported by the Special Fund of Talentdevelopment of the second affiliated hospital of Xi'an Jiaotong University (No.6YJ(ZD), and grants from National Natural Science Foundation of China (No. 81170209 to J.W. and No. 81100210 to J.X.).
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