Inflammation ( # 2016) DOI: 10.1007/s10753-016-0473-5

ORIGINAL ARTICLE

Murine Double Minute-2 Inhibition Attenuates Cardiac Dysfunction and Fibrosis by Modulating NF-κB Pathway After Experimental Myocardial Infarction Hao Zhao,1 Ruijuan Shen,2,3 Xiaobin Dong,1 and Yi Shen2

Abstract—Inflammation has been implicated in myocardial infarction (MI). MDM2 associates with nuclear factor-κB (NF-κB)-mediated inflammation. However, the role of MDM2 in MI remains unclear. This study aimed to evaluate the impacts of MDM2 inhibition on cardiac dysfunction and fibrosis after experimental MI and the underlying mechanisms. Three-month-old male C57BL/6 mice were subjected to left anterior descending (LAD) coronary artery ligation for induction of myocardial infarction (MI). Immediately after MI induction, mice were treated with Nutlin-3a (100 mg/kg) or vehicle twice daily for 4 weeks. Survival, heart function and fibrosis were assessed. Signaling molecules were detected by Western blotting. Mouse myofibroblasts under oxygen and glucose deprivation were used for in vitro experiments. MDM2 protein expression was significantly elevated in the mouse heart after MI. Compared with vehicle-treated animals, Nutlin-3a treatment reduced the mouse mortality. Nutlin-3a treatment improved heart function and decreased the infarct scar and fibrosis compared with vehicle. Furthermore, MDM2 inhibition restored IκB and inhibited NF-κB activation, leading to suppressed production of proinflammatory cytokines in the heart after MI. The consistent results were obtained in vitro. MDM2 inhibition reduced cardiac dysfunction and fibrosis after MI. These effects of MDM2 inhibition is mediated through modulating NF-κB activation, resulting in inhibition of inflammatory response. KEY WORDS: myocardial infarction; fibrosis; MDM2; NF-κB; inflammation.

INTRODUCTION The intense inflammatory response, triggered by MI, is essential for cardiac repair, but in the meanwhile implicated in the pathogenesis of post-MI heart dysfunction and remodeling [1]. Unfortunately, because of the close relationships between inflammation and repair, non-selective inhibition of inflammation after MI may confer adverse effects on repair, scar formation, and remodeling [2]. Thus, 1

Emergency Department, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, 250014, China 2 Department of Health, Qingdao Municipal Hospital, No.5 Donghaizhong Road, Qingdao, 266071, Shandong, China 3 To whom correspondence should be addressed at Department of Health, Qingdao Municipal Hospital, No.5 Donghaizhong Road, Qingdao, 266071, Shandong, China. E-mail: [email protected]

selective targeting of injurious proinflammatory mediators or signals is needed. Murine double minute-2 (MDM2) is an intracellular protein with multiple biologic functions. It was first described to limit p53-mediated cell cycle arrest and is considered as a potential therapeutic target in cancer therapy [3]. However, MDM2 has a number of p53-independent effects. For example, MDM2 has p53-independent transcription factor-like effects in nuclear factor-kappa beta (NF-κB) activation, and MDM2 promotes tissue inflammation and has adverse implications in experimental autoimmune disorders [4]. As a new entry, MDM2 was identified to act as a co-transcription factor for NF-κB at cytokine promoters. In this context, we aimed to examine the expanding role of MDM2 as a regulator of NF-κB signaling and its potential impact on myocardial remodeling and

0360-3997/16/0000-0001/0 # 2016 Springer Science+Business Media New York

Zhao, Shen, Dong, and Shen cardiac function following experimental MI and the underlying mechanisms in the present study.

MATERIALS AND METHODS

heart rate stabilized at 400 to 500 beats per minute. Twodimensional short-axis images were obtained using a high resolution Micro-Ultrasound system (Vevo 770, VisualSonics Inc., Toronto, Canada, 40-MHz probe) as described [7]. Following echocardiography, the heart tissue was harvested.

Induction of Myocardial Infarction in Mice All procedures were approved by the Shandong University of Traditional Chinese Medicine Institutional Animal Care and Use Committee, in line with the NIH Guide for the Care and Use of Laboratory Animals. The 3-monthold male C57BL/6 mice (Jackson Laboratory, Bar Harbor, USA) were fed ad libitum with a normal chow diet. The mice underwent left anterior descending coronary artery (LAD) occlusion for the induction of myocardial infarction. In brief, the mice were exposed to inhaled 1.5% isofluorane. The heart was pushed out of the thorax with direct visual control, and the LAD was sutured and ligated using a prolene suture [5]. The heart was immediately placed back into the intrathoracic space after the knot was tied, followed by manual evacuation of the pneumothorax and closure of muscle and the skin using the previously placed purse-string suture. Regional ischemia was confirmed by visual inspection (color change). Shamoperated control mice underwent the same surgical procedure, but without occlusion. Nutlin-3a Treatment In Vivo After the induction of MI, mice received Nutlin-3a (Alexis Biochemicals, San Diego, CA) at a dose of 100 mg/kg twice daily for 4 weeks (n = 20 mice) [6]. The control animals (n = 20 mice) were treated with vehicle (1% Klucel, 0.1% Tween 80) alone. All mice were killed by cervical dislocation at the end of the fourth week of treatment, and tissues and plasma were harvested for a comparative analysis. In a second independent experiment, mouse survival was assessed between different groups (n = 20 mice/group). Assessment of Cardiac Function by Echocardiography To measure cardiac function, two-dimensional transthoracic echocardiography was performed in mice. Measurements of LV end-diastolic and end systolic diameters (LVEDD and LVESD) and wall thickness and LV ejection fraction (LVEF) and fractional shortening (LVFS) were taken from the myocardium proximal to the infarct. Mice were lightly anesthetized with 1.5% isofluorane until the

Plasma Brain Natriuretic Peptide Plasma brain natriuretic peptide (BNP) was determined with commercially available ELISA Kit (RayBiotech, Phoenix Pharmaceuticals Inc., USA). Histology Heart tissues were analyzed for scar size and fibrosis by Masson’s Trichrome staining 4 weeks after MI, as described previously [8]. The fibrosis area was calculated as the ratio of the length of fibrotic area to the length of LV inner circumference using a computerized digital image analysis system (Image-Pro Plus 6.0). Hypoxic Treatment of Mouse Myofibroblasts and MDM2 Knockdown Myofibroblasts were isolated from 1-day-old neonatal C57BL/6J mice as reported previously [9]. Briefly, left ventricle tissue was excised, washed with PBS, and cut into pieces of 1∼2 mm. Enzymatic digestion using collagenase type II (100 U) and 0.125% trypsin-EDTA was employed to dissociate the tissue pieces, which were agitated at 37 °C for 60 min. Supernatant was collected every 10 min into a conical tube and digestion was neutralized with 1/10 volume of fetal bovine serum. Finally, the cells were centrifuged at 1200 rpm for 10 min, resuspended in fresh medium (DMEM containing 10% FBS), and plated into 25 cm2 culture flasks. Non-adherent cells were removed after 60 min. The remaining cells were propagated 3∼5 passages, which spontaneously differentiated into myofibroblasts. The myofibroblasts were plated on dishes and incubated in DMEM containing 10% FBS at 37 °C for 24 h. The cells were transfected with MDM2 siRNA using Lipofectamine 2000 (Invitrogen; Carlsbad, CA) and incubated for 48 h. Control (scrambled) siRNA was obtained from Santa Cruz, USA. The siRNA targeting MDM2 sense and antisense strands were GCUUCGGAACAAGA GACCC and GGGUCUCUUGUUCCGAAGC (Santa Cruz; USA). Then, the myofibroblast culture medium was replaced with glucose- and serum-free DMEM for oxygen and glucose deprivation, and the cells were kept in an airtight

MDM2 Inhibition Attenuates Myocardial Infarction anoxia chamber saturated with 95% N2/5% CO2. After 2 or 4 h of incubation under oxygen and glucose deprivation, proteins were extracted from myofibroblasts and subjected to Western blot and ELISA. Western Blot The heart tissues in non-infarcted areas or cells were prepared using cell lysis buffer (Cell Signaling Technology) and homogenized in the presence of proteinase inhibitors and protein concentrations evaluated by the DC protein assay (Bio-Rad, Hercules, CA, USA). The homogenates were cleared by centrifugation at 14,000×g for 20 min; the protein concentration was determined by Bradford analysis (Bio-Rad, CA), and the samples were normalized by protein content. Equal amounts of protein were electrophoresed on a 12% SDS–PAGE gel, transferred to a nitrocellulose membrane. The membranes were probed with anti-MDM2, IκBα, NF-κB p65, GAPDH, and Histone 2A (Cell Signaling, Danvers, MA), followed by incubation with horseradish peroxidase (HRP)-coupled antirabbit secondary antibody (Cell Signaling Technology, Beverly, MA). X-ray film was used for photon detection and image development. The relative density of the bands on the film was determined by ImageJ software. ELISA The concentrations of TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) from the mouse heart in non-infarcted areas and cell supernatants were measured by quantitative sandwich enzyme immunoassay (ELISA, R&D Systems, USA) according to the manufacturer’s instructions. Statistical Analysis All values are expressed as mean ± SD. Differences between two groups were analyzed via the Student t test, and differences between three groups were evaluated via one-way ANOVA. A p value of less than 0.01 was considered significant.

Fig. 1. Dynamics of MDM2 expression in mouse heart after myocardial infarction (MI). MDM2 expression was upregulated 24 h after MI surgery and continued to be overexpressed for at least 4 weeks after MI as shown with representative Western blot (top panel) and semi-quantification (bottom panel). n = 10 mice/group *p < 0.01, compared with sham-operated group.

at 24 h after MI and remained elevated at weeks 1 and 4 post MI. This showed that MDM2 protein expression was induced by MI in the mouse heart. Nutlin-3a Significantly Improved the Survival Rate After MI To test whether MDM2 inhibitor Nutlin-3a increased the survival of mice after MI, mice with established MI were either treated orally with vehicle or Nutlin-3a (100 mg/kg twice daily). As shown in Fig. 2, Kaplan– Meier survival study indicated that the mortality rate in mice treated with Nutlin-3a was significantly reduced after MI.

RESULTS MDM2 Expression Was Upregulated in the Heart After MI First of all, we detected the time course of MDM2 protein expression. As shown in Fig. 1, compared with sham group, MDM2 was increased by more than threefold

Nutlin-3a Attenuated Cardiac Dysfunction, Fibrosis, and Infarct Size After MI We examined whether blockade of MDM2 improved LV systolic function after MI. EF was reduced significantly more (p < 0.05) in the vehicle-treated group at 4 weeks after MI than in the Nutlin-3a group (Table 1). FS was also

Zhao, Shen, Dong, and Shen Nutlin-3a Inhibited NF-κB Activation and Inflammation in the MI Heart

Fig. 2. Survival rate at 4 weeks after myocardial infarction (MI). Three independent groups (n = 20 mice/group) were used for evaluated the survival. Kaplan–Meier analysis showed significantly lower mortality in Nutlin-3a-treated MI mice than in vehicle-treated MI mice (log-rank p = 0.013).

significantly improved in the Nutlin-3a-treated group compared with the vehicle-treated group after 4 week MI (Table 1). BNP is considered a gold standard biomarker in determining the diagnosis and prognosis of heart failure [10]. We probed BNP plasma levels and found that vehicle treatment yielded significantly higher BNP titers post-MI. Nutlin-3a treatment kept the BNP level comparable to that of sham-operated animals (Fig. 3a). We then explored the effects of Nutlin-3a on the extent of fibrosis after MI by Masson’s trichrome staining. As illustrated in Fig. 3b, fibrosis was observed in the hearts of vehicle-treated MI group, and this fibrosis was significantly reduced by Nutlin-3a treatment, as indicated by the semi-quantitative measurement (49.38 ± 4.69 vs 14.12 ± 4.09%, p < 0.01).

To gain insights into the related signaling pathways, we focused on NF-κB pathway because MDM2 has been shown to induce expression of the p65 subunit of NF-κB, which is key pathway in inflammation [11, 12]. A significant reduction of IκBα protein was observed in the vehicle-treated MI heart; however, its level was restored in the heart of Nutlin-3a-treated mice after MI (Fig. 4). This result suggests that MDM2 mediates IκB degradation in the MI heart. Degradation of IκB frees NF-κB dimers, which in turn translocate to the nucleus and promote transcription. Consistently, the protein levels of p65 protein, indicative of NF-κB activity, in the nucleus were significantly increased in the vehicle-treated MI heart, which was reduced in the Nutlin-3a-treated MI heart (Fig. 4). Furthermore, we examined the expression of NF-κBtargeted cytokines, such as TNF-α, IL-1β, and MCP-1 in non-infarcted areas. We found that TNF-α, IL-1β, and MCP-1 were significantly increased in the vehicle-treated MI heart (Fig. 5). By contrast, Nutlin-3a-treated MI heart showed significantly lower levels of these cytokines.

MDM2 Silencing Inhibited NF-κB-Mediated Inflammation in the Mouse Myofibroblasts To further confirm the mechanism of MDM2 inhibition, we used the MDM2siRNA to treat the mouse myofibroblasts under oxygen and glucose deprivation. Western blot analysis showed that the expression of IκBα was significantly elevated, and NF-κBp65 was reduced 2 or 4 h after MDM2siRNA treatment in cultured myofibroblasts under oxygen and glucose deprivation (Fig. 6a–c). ELISA revealed that the levels of TNF-α, IL1β, MCP-1 and were significantly reduced by MDM2siRNA after 2 or 4 h treatment in cultured myofibroblasts when compared to the control (Fig. 6d–f).

Table 1. Echocardiographic Data at MI After 4 Weeks Parameter

Sham

Vehicle

Nutlin-3a

LV diastolic dimension (mm) Ejection fraction (%) Shortening fraction (%) Heart rate (bpm)

2.60 ± 0.08 75.16 ± 3.25 51.88 ± 2.12 436 ± 7

5.10 ± 0.04* 27.54 ± 3.87* 18.61 ± 2.89* 415 ± 14

5.24 ± 0.50# 30.64 ± 3.52# 27.18 ± 4.31# 391 ± 16*

Values are mean ± SD; n = 8–10 animals/group. Animals were allotted to the following three groups: (1) sham operated (sham), (2) myocardial infarction (MI) + vehicle, and (3) MI + Nutlin-3a LV left ventricular *p < 0.01 vs. sham, #p < 0.01 vs. vehicle by one way ANOVA

MDM2 Inhibition Attenuates Myocardial Infarction

Fig. 4. Effect of Nutlin-3a treatment on IκB degradation and NF-κB signaling after MI. Nutlin-3a restored IκBα protein and reduced NF-κB activity in the vehicle-treated MI heart. n = 10 per group; *p < 0.01 vs vehicle. The upper two panels are representative Western blots for IκB protein from 10 different hearts in each group. The lower panel is the quantification of IκB protein relative to GAPDH and p65 protein relative to histone 2A. Data are mean ± SD, n = 6–8 mice per group. *p < 0.05 vs sham group; #, p < 0.05 vs *p < 0.01 vs vehicle-treated group.

Fig. 3. Effect of Nutlin-3a on cardiac function and reduced fibrosis after MI. a Plasma brain natriuretic peptide (BNP) was determined with commercially available ELISA Kit in Nutlin-3a-treated MI mice than in vehicle-treated MI mice 4 weeks afterwards. b Fibrosis was evaluated in Masson’s Trichrome-stained heart sections from mice sacrificed 4 weeks after MI, magnitude ×200; c Fibrosis quantified as the ratio of the length of fibrotic area to the length of LV inner circumference and reported as a percentage. n = 6–8 mice per group; *p < 0.01 vs vehicle-treated group.

DISCUSSION Although MDM2 has been implicated in the heart development [13], it remains unclear about the role of MDM2 in the MI. To the best of our knowledge, this is the first paper that evaluates the potential of MDM2 as a therapeutic target in the ischemic myocardium following MI. Because aberrant sterile inflammation plays a crucial

Fig. 5. Effect of Nutlin-3a treatment on inflammation after MI. The levels of TNF-α, IL-1β, and MCP-1 were measured in non-infarcted areas 4 weeks after MI. Data are mean ± SD, n = 6–8. *p < 0.01 vs vehicletreated group.

Zhao, Shen, Dong, and Shen

Fig. 6. Effect of MDM2 silencing on NF-κB-mediated inflammation in mouse myofibroblasts. We used the MDM2siRNA to treat the mouse myofibroblasts under oxygen and glucose deprivation. The expression of IκBα was significantly elevated, and NF-κBp65 was reduced 2 or 4 h after MDM2siRNA treatment in cultured myofibroblasts under oxygen and glucose deprivation (a–c). ELISA revealed that the levels of TNF-α, IL-1β, and MCP-1 were significantly reduced by MDM2siRNA after 2 or 4 h treatment (d–f). Data are mean ± SD, n = 6–8. *p < 0.01, #p < 0.05 vs their respective controls.

role in MI, we hypothesized that MDM2 blockade with Nutlin-3a might protect from inflammation-related myocardial damage following MI. First of all, we detected the protein expression of MDM2 in MI heart. We found that MDM2 was increased by more than threefold at 24 h after MI and remained elevated at weeks 1 and 4 post MI. This confirmed that MDM2 protein expression was induced by MI in the mouse heart. Furthermore, we explored the therapeutic value of MDM2 blockade in experimental MI. As expected, our data indicate that MDM2 inhibition blocked the induction of proinflammatory cytokines and improved cardiac function and fibrosis in the heart during MI. MDM2 is a transcriptional target of both NF-κB and p53 signaling, linking inflammation, and carcinogenesis [12, 14]. In contrast, MDM2 is also a regulator of both NF-κB and p53 pathways and can be proinflammatory and pro-proliferative or anti-inflammatory and anti-survival [4, 12, 15]. MDM2 also has a p53-independent function in NF-κB signaling, which facilitates post-ischemic inflammation and increases extent of acute tubular injury [16]. In the same manner, MDM2 inhibition prevents glomerular inflammation and podocyte loss in early adriamycin nephropathy [17]. In a lupus nephritis model, MDM2 blockade by Nutlin-3a leads to inhibition of most autoreactive T cells and plasma cells and consequently to the suppression of intrarenal and systemic inflammation [18]. MDM2

blockade with nutlin also abrogates LPS-induced lung inflammation due to impaired NF-κB DNA binding in neutrophils and macrophages [19]. Furthermore, MDM2 blockade with Nutlin-3a inhibits LPS-induced lung inflammation or attenuates atherosclerosis by suppressing NFκB-dependent inflammation in vascular smooth muscle cells [20, 21]. Cytokines and chemokines play crucial roles in stimulation of the postinfarction inflammatory response and in the pathogenesis of cardiac remodeling. TNF-α is crucially involved in the pathogenesis and progression of atherosclerosis, myocardial ischemia/reperfusion (MI/R) injury, and heart failure [22]. It is reported that neutralization of TNF-α is able to limit infarct size and to reduce early LV diastolic dysfunction in rats after MI [23]. TNF-α inhibitor depresses oxidative/nitrative stress to attenuate myocardial injury in I/R [24]. In experimental models of myocardial infarction, IL-1β synthesis is markedly upregulated after infarction [25]. Deficiency of Interleukin-1 receptor type I attenuates dilation of the infarcted heart and adverse remodeling [26]. Treatment with anti-IL-1β antibodies exerted protective actions in a rat model of myocardial and in a mouse model of nonreperfused infarction [27, 28]. Pilot studies have shown the good safety and effects of IL-1 blockade with anakinra on left ventricular (LV) remodeling after acute myocardial infarction [29]. In addition, the CC chemokine MCP-1/CCL2 is rapidly

MDM2 Inhibition Attenuates Myocardial Infarction upregulated in the infarcted myocardium [30] and genetic deletion of MCP-1 or its receptor CCR2 attenuated adverse remodeling after MI, inhibiting recruitment of proinflammatory monocytes, and decreasing cytokine expression in the infarct [31, 32]. In a model of nonreperfused infarction, anti-MCP-1 therapy exerted beneficial actions on the infarcted ventricle, reducing mortality, attenuating chamber dilation, and improving systolic function [33]. These data suggest that MCP-1/CCL2 may be a promising therapeutic target after myocardial infarction. Results from this study indicate that MDM2 inhibition suppressed production of proinflammatory cytokines in the heart after MI. Taken together, inhibition of MDM2 preserved LV function and inhibited fibrosis by targeting NF-κB-mediated inflammation after chronic MI. Since MDM2 increases naturally during chronic MI, it could play a role in promoting inflammatory response with chronic myocardial ischemia, and conversely, blocking MDM2 could be a potential novel therapeutic target for chronic myocardial ischemic disease.

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COMPLIANCE WITH ETHICAL STANDARDS

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All procedures were approved by the Shandong University of Traditional Chinese Medicine Institutional Animal Care and Use Committee, in line with the NIH Guide for the Care and Use of Laboratory Animals.

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Conflict of Interest. The authors declare that they have no conflict of interest.

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