anti-inflammatory profile of levosimendan in cecal ... · was associated with inhibition of the rho...

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e508 www.ccmjournal.org November2015•Volume43•Number11

Objectives:Thecalciumsensitizerlevosimendanisusedintreat-mentofdecompensatedheart failureandmayalsoexhibit anti-inflammatory properties. We examined whether treatment withlevosimendan is substantially beneficial inmicewith cecal liga-tionandpuncture–inducedpolymicrobialsepsis,and itsarbitra-tionmechanismwasexploredinthemousemacrophagecelllineRAW264.7.Design:Laboratoryandanimal/cellresearch.Setting:Universityresearchlaboratory.Subjects:BALB/cmice (8–10wkold) andmousemacrophagecelllineRAW264.7cells.Interventions: Levosimendan (0.5μg/kg/min)wasadministeredtomice through an osmotic pump thatwas implanted into theperitoneal cavity immediately following surgery. In RAW264.7cells,levosimendanwasaddedtotheculturemedium30minutesbeforelipopolysaccharide.Measurements and Main Results:Whenlevosimendanwascon-tinuously administered to cecal ligation and puncture–inducedsepticmice,asignificantimprovementofleftventricularfunction

was found without any change in heart rate, and hypotensionwassignificantlymitigated.Furthermore,levosimendanconferredsubstantialprotectionagainstsepsis-associated inflammation inmice,as indicatedby reduced lung injuryanddecreasedbloodproinflammatoryandchemotacticcytokinelevels.Thesebeneficialeffectsof levosimendan ledtoasignificant improvementofsur-vivalinmiceaftercecalligationandpuncture.Inendotoxin-stim-ulated RAW264.7 macrophages, treatment with levosimendanandpimobendansuppressedoverproductionofproinflammatoryandchemotacticcytokines.Levosimendanandpimobendanwerewithout effect on activation of the nuclear factor-κB, mitogen-activatedprotein kinase, andAktpathways. Instead, levosimen-danandpimobendanpreventedhighmobilitygroupbox1releasefromthenucleustotheextracellularspaceinmacrophages.Thiswasassociatedwith inhibitionoftheRhokinasesignalingpath-way.Theelevatedserumhighmobilitygroupbox1levelsincecalligationandpuncture–inducedsepticmicewerealsoinhibitedbycontinuedadministrationoflevosimendanandpimobendan.Conclusions:Wedefineanovelmechanismfortheanti-inflamma-toryactionoflevosimendanandsuggestthatthepharmacologicalprofilesoflevosimendanasbothaninotropeandananti-inflamma-toryagentcouldcontribute to itsclinicalbenefit inpatientswithsepsiswithheartproblems.(Crit Care Med2015;43:e508–e520)Key Words:hemodynamicdysfunction;highmobilitygroupbox1; inflammation; levosimendan; macrophages; nuclear factor-κB; pimobendan;sepsis

Sepsis is considered a complex disorder arising from the dys-regulation of a systemic inflammatory response to infect-ing microorganisms, such as bacteria (1), but remains

an enigmatic, poorly understood disease (2). Sepsis alters the expression of many pathogenetic factors that can potentially give rise to abnormalities in cardiovascular, respiratory, metabolic, and coagulation systems, and severe sepsis is identified when sepsis affects major organs within the body, ultimately leading

Copyright©2015bytheSocietyofCriticalCareMedicineandWolters KluwerHealth,Inc.AllRightsReserved.

DOI: 10.1097/CCM.0000000000001269

*See also p. 2522.1DepartmentofMolecularandMedicalPharmacology,GraduateSchoolofMedicineandPharmaceuticalSciences,UniversityofToyama,Toyama,Japan.

2DepartmentofAnesthesiologyandPainReliefCenter,UniversityofTokyoHospital,Tokyo,Japan.3DepartmentofEmergencyandCriticalCareMedicine,NagoyaUniversityGraduateSchoolofMedicine,Nagoya,Japan.CurrentaddressforDr.Yokoo:DepartmentofHealthandNutritionalSci-ences,FacultyofHealthPromotionalSciences,TokohaUniversity,Hama-matsu,Japan.

Dr.Hattori’s institution receivedgrant support:Grant-in-Aids forScien-tificResearch(26460336)andGrant-in-AidsforChallengingExploratoryResearch (15K15661) from theMinistry of Education,Culture, Sports,Science,andTechnologyofJapan.Theremainingauthorshavedisclosedthattheydonothaveanypotentialconflictsofinterest.

Forinformationregardingthisarticle,E-mail:[email protected]

Anti-Inflammatory Profile of Levosimendan in Cecal Ligation-Induced Septic Mice and in Lipopolysaccharide-Stimulated Macrophages*

Qiang Wang, MD, PhD1; Hiroki Yokoo, MD, PhD1; Michinori Takashina, MS1;

Kimimasa Sakata, MD, PhD1; Wakana Ohashi, PhD1; Lobna A. Abedelzaher, MD1;

Takahiro Imaizumi, BS1; Takuya Sakamoto, BS1; Kohshi Hattori, MD2; Naoyuki Matsuda, MD, PhD3;

Yuichi Hattori, MD, PhD1

mailto:[email protected]

Copyright © 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.

Online Laboratory Investigations

CriticalCareMedicine www.ccmjournal.org e509

to one or more organ failure (3). Septic shock is associated with hypotension requiring vasopressor support despite adequate fluid resuscitations in order to maintain vascular perfusion and results in disruption of the organ oxygen supply-demand bal-ance, which also eventually contribute to the development of multiple organ failure (4). Severe sepsis and septic shock are major causes of death in ICUs (5). The number of patients with severe sepsis and septic shock continues to rise because of the gain in life expectancy and the increase in the number of immunocompromised patients, among other reasons (6). The great mortality of severe sepsis and septic shock has intensified research seeking new strategies for the disease therapy.

Calcium-sensitizing agents are cardiac inotropes that have attracted growing interest over the nearly 30 years (7, 8). They act as a direct activator of motor proteins such as myosin, an enhancer of force generated by a cross-bridge or an agent that augments Ca2+-troponin C by a cross-bridge (8, 9). Levosimendan is the most widely studied calcium sensitizer to date and is clini-cally used against decompensated heart failure (9–11). In a few limited trials and case series, some promising data indicate that levosimendan may specifically offset sepsis-associated cardiac dysfunction and thus improve hemodynamics (12, 13). In this regard, several clinical trials of levosimendan in patients with septic shock have been run in a proactive way. Interestingly, available evidence suggests that levosimendan may also have anti-inflammatory properties (14, 15). These composite actions of levosimendan raise the theoretical possibility that this agent may have value as a treatment of sepsis. Indeed, some benefits of levosimendan have been shown in different experimental animal models with septic shock (16–18). However, the information on a set of the underlying mechanisms by which levosimendan may have a favorable impact on sepsis episodes is very limited.

Herein, we examined whether treatment with levosimendan is substantially beneficial in mice with cecal ligation and punc-ture (CLP)-induced polymicrobial sepsis. CLP-induced sepsis is an animal model that has high relevance to humans because it reproduces many hallmarks of sepsis that occur in patients (19). Further studies were then undertaken using the mouse macrophage cell line RAW264.7 to gain insight into possible mechanism(s) involved in the ability of levosimendan to mitigate sepsis. In a series of in vitro experiments, pimobendan, which is approved for use in heart failure patients only in Japan (20), was also used as another pyridazinone derivative calcium sensitizer.

MATERIALS AND METHODS

Animal Models of Sepsis and Drug AdministrationAll animal studies were conducted in accordance with the National Institute of Health Guidelines on the use of labora-tory animal and with approval of the Care and Use Committee of the University of Toyama. The surgical procedure to gener-ate CLP-induced sepsis was performed as previously described (21–23). In brief, male BALB/c mice, 8–10 weeks old, were anesthetized with 3–4% sevoflurane, and a middle abdomi-nal incision was made. The cecum was mobilized, ligated, and punctured twice with a 21-gauge needle, allowing exposure of

faces, the bowel was repositioned, and the abdomen was closed with sterile suture. In the levosimendan-treated CLP group, microosmotic pumps (Alzet, model 1003D; Durect, Cuper-tino, CA) delivering levosimendan (0.5 μg/kg/min) were placed into the peritoneal cavity before the abdomen was closed. For point of reference, the human levosimendan IV dosage is 0.2 μg/kg/min (24). Levosimendan was dissolved in a small amount of dimethyl sulfoxide and then diluted in a 5% (w/v) glucose solution. The placebo group received an equal volume of 5% glucose (100 μL) by the osmotic pump. Sham-operated con-trol mice underwent the same procedure except for ligation and puncture of the cecum. We used a noninvasive computerized tail-cuff system for measuring blood pressure in mice (22, 25).

Cell CultureRAW264.7 cells, a mouse macrophage cell line, were purchased from RIKEN BRC (Tsukuba, Japan) and maintained in 10-cm dishes with Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% Co

2 atmosphere. Cells in passages

8–12 were used in the experiments. When cells were treated with levosimendan or pimobendan, these agents were added 30 min-utes before the challenge with lipopolysaccharide (LPS; Esch-erichia coli 055:B5; List Biological Laboratories, Campbell, CA).

Isolation of Peritoneal MacrophagesFive- to 8-week-old male BALB/c mice were injected intra-peritoneally with 2 mL of 3% thioglycollate broth. Closed peritoneal lavage was performed using 10 mL ice-cold sterile phosphate buffer solution (PBS). Peritoneal exudate cells were collected and washed once with DMEM supplemented with 10% FBS. A couple of hours later, nonadherent contaminating cells were removed by washing with PBS. Peritoneal macro-phages were cultured in DMEM overnight at 37°C in a humidi-fied incubator containing 5% Co

2 before starting experiment.

Echocardiographic MeasurementsEchocardiographic studies were carried out using an ultra-sound animal-imaging system (Prospect; S-Sharp, Taipei, Taiwan) at a frequency of 40 MHz with a transducer (acous-tic pressure, 0.59 MPa; MI = 0.09) with a diameter of 7 mm and a fixed focus at 12 mm. The left ventricular dimensions in diastole (LVDd) and in systole (LVDs) and percent fractional shortening (FS) were estimated using the M-mode measure-ments. Ejection fraction (EF) was calculated from derived vol-umes, which are computed based on the “Teicholz” equations.

Histologic ExaminationFor routine histology, inflation-fixed lungs were harvested, fixed, dehydrated, paraffin-embedded, and sliced into 4-μm-thick sections (22, 23, 25). After deparaffinization, slides were stained with hematoxylin and eosin using standard methods. A semiquantitative morphometric analysis of lung injury was performed by scoring from 0 to 4 (none, light, moderate, severe, very severe) for the following categories: neutrophil

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Wang et al


infiltration, pulmonary edema, and disorganization of lung parenchyma and hemorrhage (22, 23). A total lung injury score was calculated by adding the individual scores in every animal and averaging the total values in each group. For apoptotic cell detection, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) was performed using ApopTag Plus Peroxidase In Situ Apoptotic Detection Kit (Mil-lipore, Billerica, MA) according to the manufacturer’s instruc-tions. Additional details are described elsewhere (23).

Enzyme Immunoassay for CytokinesBlood and culture medium levels of tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, and monocyte chemoattrac-tant protein (MCP)-1 were measured by the use of commer-cially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manu-facturer’s instructions. To measure the concentrations of high mobility group box (HMGB) 1 in serum or culture medium samples, HMGB1 Detection Kit (Chondrex, Redmond, WA) was used. The plate was read on a microplate reader (Nippon-InterMed, Tokyo, Japan). Assays were performed in duplicate.

RNA Extraction and Quantitative Real-Time Polymerase Chain ReactionTotal RNA was isolated from cells or lung tissues with Sepazol-RNA I Super G (Nacalai). PrimeScript RT Master Mix (Takara Bio, Ohtsu, Japan) or ReverTra Ace quantitative real-time poly-merase chain reaction (qPCR) Master Mix (Toyobo, Osaka, Japan) was used for the reverse transcription reaction, and real-time PCR analyses were performed using SYBR Premix Ex Taq II (Takara Bio) or THUNDERBARD SYBR qPCR Mix (Toyobo). The TNF primer sequences were 5′-GTTCTATG-GCCCAGACCCTCAC-3′ (sense) and 5′-GGCACCACTAGTTG-GTTGTCTTTG-3′ (antisense), the IL-1β primer sequences were 5′-TCCAGGATGAGGACATGAGCAC-3′ (sense) and 5′-GAAC-GTCACACACCAGCAGGTTA-3′ (antisense), the IL-6 primer sequences were 5′-CCACTTCACAAGTCGGAGGCTTA-3′ (sense) and 5′-GCAAGTGCATCATCGTTGTTCATAC-3′ (antisense), the MCP-1 primer sequences were 5′-GCATC-CACGTGTTGGCTCA-3′ (sense) and 5′-CTCCAGCCTACT-CATTGGGATCA-3′ (antisense), and the HMGB1 primer sequences were 5′-AGCCCTGTCCTGGTGGTATTTTCAA-3′ (sense) and 5′-GCTGTGCACCAACAAGAACCTGC-3′ (anti-sense). The internal standard used was the ubiquitously expressed housekeeping gene glyceraldehyde-3-phosphate dehy-drogenase (GAPDH), which was determined with the primer pair (sense, 5′-TGTGTCCGTCGTGGATCTGA-3′; antisense, 5′-TTGCTGTTGAAGTCGCAGGAG-3′).

Western Blot AnalysisBlotting procedure, chemiluminescent detection, and densi-tometric analysis were performed as previously described by our laboratory (26). Cells were harvested and lysed in 300 μL of radio-immunoprecipitation assay buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 7.4; Thermo, Rockford, IL)

containing protease inhibitor cocktail on ice. The lysates were centrifuged at 18,000 × g for 10 minutes at 4°C, and the result-ing supernatants were collected. The proteins in the superna-tant were measured using BCA Protein Assay Kit (Thermo). When required, nuclear protein extracts from macrophages were prepared as described previously (27). Total supernatants (10–30 μg of protein) were run on 12% or 14% polyacryl-amide gel and electrotransferred onto polyvinylidene fluoride filter membrane. The membrane was blocked for 60 minutes at room temperature in Odyssey blocking buffer, followed by overnight incubation with primary antibody at 4°C. The mem-brane was washed four times with Tween-phosphate-buffered

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Figure 1. Effect of levosimendan (Levo) treatment on mortality, hypotension, and heart rate in mice with cecal ligation and puncture (CLP)-induced sepsis. A, Kaplan-Meier survival curves. No deaths occurred in mice that underwent sham procedure (n = 8). Levosimendan was continuously given during the first 3 d after CLP. Twelve and 10 animals were used for untreated and levosimendan-treated CLP groups, respectively. All animals were given a subcutaneous injection of 600 μL of saline after the surgery. Mortality was observed over 5 d. B, Mean arterial blood pressure before and 6 and 12 hr after surgery. The data are presented as mean ± sem (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs sham. #p < 0.05, ##p < 0.01 vs CLP alone. C, Heart rate changes 24 hr after surgery. The data are presented as mean ± sem (n = 4–8 per group). Note that there was no significant difference between each group.




saline and incubated with IR-Dye-labeled secondary antibodies at room temperature for 120 minutes in the dark. Fluorescent of Infrared-Dye was analyzed by Odyssey CLx Infrared Imaging System (LI-COR Bioscience, Lincoln, NE).

The following antibodies, which are commercially available, were used: anti-human IκBα rabbit monoclonal antibody (1:1,000 dilution; #4812; Cell Signaling, Danvers, MA), anti-human nuclear factor (NF)-κB rabbit monoclonal antibody (1:1,000; #3034; Cell Signaling), anti-human extracellular signal-regulated protein kinase (ERK) 1/2 mouse monoclonal antibody (1:1,000; #4696; Cell Signaling), anti-human phospho-ERK1/2 (Thr-202/Tyr-204) rabbit monoclonal antibody (1:1,000; #4370; Cell Signaling), anti-human c-Jun N-terminal kinase (JNK) rabbit monoclonal antibody (1:1,000; #9258; Cell Signaling), anti-human phospho-stress-acti-vated protein kinase/JNK (Thr-183/Tyr-185) mouse monoclonal

antibody (1:1,000; #9255; Cell Signaling), anti-human p38 rab-bit monoclonal antibody (1:1,000; #8690; Cell Signaling), anti-human phospho-p38 (Thr-183/Tyr-182) mouse monoclonal antibody (1:1,000; #9216; Cell Signaling), anti-human Akt rabbit polyclonal antibody (1:2,000; #4691; Cell Signaling), anti-human phospho-Akt (Ser-473) rabbit polyclonal antibody (1:1,000; #4060; Cell Signaling), anti-human HMGB1 rabbit polyclonal antibody (1:1,000; #3935; Cell Signaling), anti-human GAPDH chicken polyclonal antibody (1:2,000–4,000; #AB2302, Millipore), anti-human Ras homolog gene family, member A (RhoA) mouse mono-clonal antibody (1:500; #ARH04, Cytoskeleton, Denver, CO), and anti-human lamin B goat poly-clonal antibody (1:200; #sc-6216; Santa Cruz Biotechnology, Santa Cruz, CA).

NF-κB Luciferase AssayRAW264.7 cells cultured in 96-well plates were cotransfected with 0.3 μg of pGL4.32-NF-κB-Luc plasmid DNA (encod-ing firefly luciferase; Promega, Madison, WI) and pGL4.74-hRluc/TK plasmid DNA (0.1 μg) (encoding Renilla lucifer-ase; Promega) using the Lipo-fectamine LTX reagent (Life Technologies, Tokyo, Japan) in accordance with the manufac-turer’s protocol. After 24 hours,

the cells were treated with LPS for 6 hours. Then, the cells were lysed and assayed for luciferase activity using the Dual-luciferase Reporter assay system (Promega) in a Lumat LB9507 luminom-eter (Berthold, Bad Wildbad, Germany).

RhoA Activation AssayThe levels of active guanine-5'-triphosphate (GTP)-bound RhoA were determined by pull-down GTP-bound RhoA with glutathione S-transferase (GST)-Rhotekin-Rho binding domain (RBD) coupled to colored glutathione-sepharose beads (Cytoskeleton). RAW264.7 cells were stimulated with 1 μg/mL LPS for 5 minutes and lysed with lysis buffer (Cytoskeleton). Lysates were centrifuged, and supernatants were incubated with beads coupled to GST-Rhotekin-RBD for 3 hours at 4°C. Beads were then washed with wash buffer (Cytoskeleton), and

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Figure 2. Transthoracic echocardiography assessment of the effect of levosimendan (Levo) treatment on left ventricular (LV) dysfunction in mice with cecal ligation and puncture (CLP)-induced sepsis. Echocardiographic measurements were performed 24 hr after CLP. A, Representative echocardiographic images for each of the three experimental groups. B–E, The averaged data of LV dimensions in diastole (LVDd), LV dimensions in systole (LVDs), percent fractional shortening (FS), and ejection fraction (EF) in the three groups are presented as mean ± sem (n = 5 per group). *p < 0.05, ***p < 0.001 vs sham. #p < 0.05, ##p < 0.01, ###p < 0.001 vs CLP alone.


Wang et al


GTP-bound RhoA was eluted with the sample buffer. Amounts of active RhoA were determined by Western blot analysis.

Statistical AnalysisNumerical data are expressed as mean ± sem. Statistical assessment of the data was made by a repeated-measure one-way analysis of variance followed by Tukey multiple comparison test. The Gehan-Breslow-Wilcoxon test was used for comparing the survival curves. A difference in the changes in blood pressure between the groups was assessed by carrying out an F test followed by a t test. The statistical significance was set at p value of less than 0.05.

RESULTS

Animal Survival, Hypotension, and Cardiac Performance After CLPWe initially examined whether levosimendan admin-istration can confer a survival advantage to mice with

CLP-induced polymicrobial sepsis. Approximately 70% of the animals subjected to CLP surgery died throughout the 5 days of observation. The ani-mals, when continuously given levosimendan immediately after CLP, displayed a survival benefit. Thus, Kaplan-Meier survival analysis showed a statistically significant differ-ence (p = 0.0342) between the two groups (Fig. 1A). A sig-nificant fall in mean arterial blood pressure was found in the animals subjected to CLP. Levosimendan administration significantly mitigated CLP-induced hypotension (Fig. 1B). There was no significant differ-ence in heart rate between the three groups (Fig. 1C).

As presented in the typical tracing images (Fig. 2A), echo-cardiography demonstrated that the LVDd was prone to decrease without changing the LVDs in CLP-induced septic mice, in comparison with sham-operated control mice (Fig. 2, B and C). Consequently, septic mice had left ventricular (LV) dysfunction as shown by significant reduc-tions in LV FS and EF (Fig. 2, D and E). Treatment of septic ani-mals with levosimendan did not alter the fall in LV diastolic per-formance but caused a significant shortening in the LVDs, resulting

in significantly improved cardiac function as measured by LV FS and EF.

Cytokine Levels and Lung Histopathology in CLP MiceWhen blood levels of proinflammatory or chemotactic cyto-kines were measured using ELISA, the sham-operated control mice had very low levels of the cytokines examined here. After induction of sepsis by CLP, marked elevations in TNF, IL-1β, IL-6, and MCP-1 were observed. Treatment with levosimendan markedly inhibited their elevated blood levels (Fig. 3A). We also examined changes in the levels of messenger RNA (mRNA) of these cytokines in lung tissues using real-time PCR. In lungs from septic mice, mRNA expression levels of TNF, IL-1β, IL-6, and MCP-1 were greatly increased. The increases in mRNA expression of these cytokines were obviously suppressed when levosimendan was given to septic mice (Fig. 3B).

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Figure 3. Effect of levosimendan (Levo) treatment on up-regulated cytokines, tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein (MCP)-1 in mice with cecal ligation and puncture (CLP)-induced sepsis. A, Blood levels of cytokines. The blood was collected 24 hr after surgery (n = 8 per group). B, Transcription levels of cytokines in lungs. Tissues were harvested 12 hr after surgery (n = 8 per group). The messenger RNA (mRNA) levels were quantified by real-time PCR. The values were expressed as a fold increase above control (sham operation) normalized glyceraldehyde-3-phosphate dehydrogenase. The data are presented as mean ± sem. *p < 0.05, **p < 0.01 vs sham. #p < 0.05 vs CLP alone.




Histopathologic analysis with hematoxylin and eosin staining of paraffin-embedded lung sections showed that CLP-induced septic mice displayed massive infiltration of inflammatory cells, marked congestion of pulmonary edema, thickening of the alveolar septum, and erythrocytes originat-ing from ruptured capillary vessels in the lung tissues (Fig. 4A). In lungs from septic animals treated with levosimendan, these histopathologic changes were attenuated. Semiquantitative assessment using lung injury score revealed that the score was significantly lowered when septic mice received levosimendan administration (Fig. 4B). The tissue sections were labeled with an in situ TUNEL assay to detect apoptotic cells in lungs. As depicted in Figure 4C, no TUNEL-positive cells were detect-able in lung sections from sham-operated control animals. Induction of sepsis by CLP resulted in a marked appearance of TUNEL-positive cells. Morphologically, apoptotic cells were

identified to endothelial cells of capillary vessels in the alveolar septa and to epithelial type II cells (25, 28). In lungs from CLP mice given levosimendan, TUNEL-positive cells were significantly reduced (Fig. 4D), providing a protective effect of levosimendan treatment on sepsis-associated pulmonary cell apoptosis.

Inflammatory Signaling in LPS-Stimulated MacrophagesWhen LPS (1 μg/mL) was applied to mouse macrophage cell line RAW264.7 cells, gene expression levels of proinflam-matory and chemotactic mol-ecules were greatly up-regulated. Treatment with levosimendan (3–30 μM) or another pyridazi-none derivative calcium sensi-tizer pimobendan (10–100 μM) suppressed the mRNA levels of TNF, IL-1β, IL-6, and MCP-1 in a concentration-dependent man-ner (Fig. 5A). When the amounts of proinflammatory and che-motactic cytokines in culture media were measured by ELISA, LPS challenge resulted in strik-ing increases in protein levels of TNF, IL-1β, IL-6, and MCP-1 secreted by RAW264.7 cells, and their increased protein levels were greatly inhibited by levosi-mendan and pimobendan (Fig. 5B). Additional experiments showed that levosimendan was

significantly effective in inhibiting the release of these proin-flammatory and chemotactic cytokines from LPS-stimulated RAW264.7 cells even when it was added at a concentration of 30 μM 30 minutes after LPS challenge (Fig. 5C).

We also examined whether levosimendan can inhibit the LPS-induced overproduction of cytokines in primary perito-neal macrophages isolated from mice. Peritoneal macrophages stimulated with 1 μg/mL LPS showed a marked increase in the mRNA levels of TNF, IL-6, and MCP-1, which was sig-nificantly suppressed by treatment with 30 μM levosimendan (Fig. 6A). In addition, a significant ability of levosimendan to inhibit the release of these proinflammatory and chemotactic cytokines from LPS-stimulated peritoneal macrophages was observed by ELISA techniques (Fig. 6B). This indicates that the inhibition by levosimendan of LPS responses is not a cell line–specific effect.

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Figure 4. Effect of levosimendan (Levo) treatment on histopathologic changes in lungs from mice with cecal ligation and puncture (CLP)-induced sepsis. Lung tissues were harvested 24 hr after surgery. A, Lung tissue sections stained with hematoxylin and eosin. B, Semiquantitative analysis of lung tissues by lung injury score. C, Lung tissue sections by an in situ terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay. Red arrowheads indicate TUNEL-positive apoptotic cells. D, Quantification of apoptotic cells. Counts of apoptotic cells were made in the sections at a final magnification of ×200, and the average of apoptotic cell number in three high-power fields per sample was calculated. The summarized results are presented as mean ± sem of data from 4 to 5 animals per group. *p < 0.05, ***p < 0.001 vs sham. #p < 0.05, ###p < 0.001 vs CLP alone.


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Figure 5. Effects of levosimendan and pimobendan treatment on up-regulated expression of cytokines, tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein (MCP)-1 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Levosimendan (Levo) and pimobendan (Pimo) were given 30 min before LPS. A, Messenger RNA (mRNA) expression levels of TNF, IL-1β, IL-6, and MCP-1 (n = 6). Cells were exposed to 1 μg/mL LPS for 6 hr. Cytokine expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. B, Cell culture media were collected 24 hr after application of 1 μg/mL LPS, and the concentrations of TNF, IL-1β, IL-6, and MCP-1 were measured by the enzyme-linked immunosorbent assay (ELISA) (n = 4). C, Levosimendan (30 μM) was added 30 min after LPS, and then TNF, IL-1β, IL-6, and MCP-1 levels in cell culture media at 24 hr after LPS were assessed by ELISA (n = 3). The data are presented as mean ± sem. ***p < 0.001 vs control. #p < 0.05, ##p < 0.01, ###p < 0.001 vs LPS alone.




The NF-κB signaling pathway plays a pivotal role in regulat-ing many of key genes that mediate the inflammatory response (29, 30). We examined whether the inhibitory effects of levo-simendan and pimobendan on the production of proinflam-matory and chemotactic cytokines are associated with their impacts on the NF-κB signaling pathway. Since the activity of NF-κB is primarily regulated by interaction with its inhibi-tory protein IκBα, degradation of IκBα after stimulation of RAW264.7 cells with LPS was monitored by Western blot. A peak reduction in IκBα was observed from 15 to 30 minutes after cells were exposed to LPS. Levosimendan and pimo-bendan did not substantially alter IκBα degradation when evaluated at 15 minutes following LPS challenge (Fig. 7A). Liberation of NF-κB from degraded IκBα ultimately leads to NF-κB nuclear translocation. Stimulation of RAW264.7 cells with LPS resulted in the translocation of NF-κB p65 into the nucleus, but neither levosimendan nor pimobendan inhibited the nuclear translocation (Fig. 7B). We also examined whether treatment with levosimendan or pimobendan can affect NF-κB transcriptional activity in LPS-stimulated RAW264.7 cells. In cells transfected with a reporter construct carrying a luciferase gene under the control of an NF-κB promoter containing five

copies of an NF-κB response element, LPS stimulation for 6 hours markedly increased NF-κB transcriptional activ-ity. Treatment with levosimen-dan or pimobendan tended to inhibit LPS-induced NF-κB activation, but their effects were not significant (Fig. 7C).

The mitogen-activated pro-tein kinase (MAPK) cascade is the key downstream pathway for LPS-stimulated signaling events (31). Hence, we tested whether the suppression of proinflammatory and chemo-tactic cytokines is regulated through the MAPK pathways. The MAPK family involves three major subgroups, includ-ing ERK1/2, JNK, and p38. When activation of ERK1/2, JNK, and p38 was assessed by their phosphorylation lev-els, LPS strongly activated all three families of MAPKs in RAW264.7 cells. Neither levosi-mendan nor pimobendan pre-vented LPS-induced increase in levels of phosphorylated ERK1/2, JNK, and p38 (Fig. 8).

Akt is a serine/threonine kinase that is known to play a critical role in negatively regu-

lating LPS-induced inflammation (32). Akt was strikingly acti-vated in LPS-stimulated RAW264.7 cells, as indicated by a great increase in Ser-473 phosphorylation of Akt. Levosimendan was without effect on LPS-induced Akt activation, whereas it was abrogated by treatment with pimobendan (Fig. 8).

HMGB1 is a nuclear factor that enhances transcriptional activation but also serves as an extracellular cytokine known to be a critical mediator of innate immune responses (33, 34). The HMGB1 mRNA levels were strongly down-regulated when RAW264.7 cells were exposed to LPS, which was partially but significantly reversed by treatment with levosimendan or pimobendan (Fig. 9A). On the other hand, LPS challenge caused a marked release of HMGB1 proteins from RAW264.7 cells. Treatment with levosimendan resulted in a return of LPS-induced HMGB1 release from cells toward baseline (Fig. 9B). In line with this finding, levosimendan and pimo-bendan prevented a reduction in HMGB1 protein levels in the nuclei of LPS-stimulated RAW264.7 cells (Fig. 9C). Even when levosimendan was added 30 minutes after LPS challenge, the LPS-induced reduction in nuclear HMGB1 protein levels was partially reversed (Fig. 9D). The Rho kinase inhibitor Y-27632 was also able to mitigate the LPS-induced reduction in nuclear

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Figure 6. Effect of levosimendan treatment on up-regulated expression of cytokines, tumor necrosis factor (TNF), interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 in lipopolysaccharide (LPS)-stimulated primary peritoneal macrophages. Mouse primary macrophages were isolated from one animal for each experiment. Levosimendan (Levo, 30 μM) was given 30 min before LPS. A, Messenger RNA (mRNA) expression levels of TNF-α, IL-6, and MCP-1 (n = 3). Cells were exposed to 1 μg/mL LPS for 6 hr. Cytokine expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. B, Cell culture media were collected 24 hr after application of 1 μg/mL LPS, and the concentrations of TNF, IL-6, and MCP-1 were measured by the enzyme-linked immunosorbent assay (n = 3). The data are presented as mean ± sem. ***p < 0.001 vs control. ##p < 0.01, ###p < 0.001 vs LPS alone.


Wang et al


HMGB1 protein levels (Fig. 9E). When we measured levels of GTP-bound active form of RhoA using pull-down assay with GST-Rhotekin, LPS strongly stimulated RhoA activity within 5 minutes and increased the level of active GTP-bound RhoA. This up-regulation of RhoA activity was negated by levosimen-dan (Fig. 9F). Finally, we examined serum HMGB1 levels in CLP-induced septic mice. Sepsis induction by CLP resulted in a marked elevation in serum HMGB1 in mice. Administration of levosimendan or pimobendan to CLP mice strongly sup-pressed the elevated levels of HMGB1 (Fig. 9G).

DISCUSSIONLevosimendan belongs to a new class of cardiac inotro-pic agents, calcium sensitizers, and is currently approved in

Europe and the United States for use in the management of acute decompensated heart failure, as levosimendan increases cardiac output without deleterious effects on myocardial oxy-gen consumption (35). In this context, levosimendan may represent a beneficial effect on sepsis-associated cardiac dys-function, although there is no definitive study supporting levosimendan as the best choice for septic patients with car-diac dysfunction (36). In the experiments using transthoracic echocardiography, our rodent model of CLP-induced sepsis exhibited limited LV cardiac performance as demonstrated by low EF and FS. A significant improvement of LV function without change in heart rate was observed when levosimendan was continuously administered to septic mice. This beneficial effect of levosimendan on sepsis-induced cardiac dysfunction could be, at least in part, attributed to the increased cardiac output. Consequently, levosimendan administration signifi-cantly improved hypotension in septic mice.

The results of the present study also indicate that levosi-mendan can confer substantial protection against acute lung injury in CLP-induced septic mice. The proinflammatory cytokines, TNF and IL-6, have been implicated in the patho-genesis of inflammatory lung injury, particularly under con-ditions of severe lung infection and sepsis (37). We found that when levosimendan was given to septic mice, elevated gene levels of proinflammatory and chemotactic cytokines in lungs were greatly inhibited, and histologic damage in lungs, including inflammatory infiltrate and parenchyma disorgani-zation, was significantly mitigated. In addition, levosimendan administration reduced cell apoptosis in lungs as determined by TUNEL assay. Accumulating evidence suggests that cell apoptosis plays a key role in the development of organ failure and mortality associated with sepsis, and it may be potentially detrimental in septic lung injury (38, 39). These findings are consistent with the results of other investigators, showing the beneficial effect of levosimendan on lung inflammation and apoptosis in CLP rats (18, 40). Furthermore, we demonstrated the ability of levosimendan to inhibit the increase in circulat-ing levels of proinflammatory and chemotactic cytokines after CLP-induced sepsis, implying that levosimendan administra-tion can reduce systemic inflammation in septic mice.

The anti-inflammatory property of levosimendan was con-firmed in in vitro experiments with RAW264.7 macrophages. Thus, levosimendan treatment strikingly inhibited proin-flammatory and chemotactic cytokine productions in LPS-stimulated RAW264.7 cells. The ability of levosimendan to suppress the up-regulated cytokine response to LPS was also found in primary mouse peritoneal macrophages. The NF-κB pathway plays a central part in the transcriptional regulation of expression of proinflammatory genes, including cytokines, chemokines, and adhesion molecules (41). In a myriad of stim-uli, commencing with LPS, IκBα is quickly phosphorylated, ubiquitinated, and degraded, releasing the NF-κB heterodimer, which then translocates from cytoplasm into nucleus to medi-ate the transcription of proinflammatory genes (42). However, levosimendan did not substantially modify IκBα degrada-tion, NF-κB p65 nuclear translocation, and NF-κB activity in

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Figure 7. Effects of levosimendan (Levo) and pimobendan (Pimo) treatment on kinetics of nuclear factor (NF)-κB activation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Levosimendan (30 μM) and pimobendan (100 μM) were given 30 min before application of 1 μg/mL LPS. A, Western blot analysis was performed using anti-IκBα monoclonal antibody. Effects of levosimendan and pimobendan were evaluated 15 min following LPS challenge. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was served as loading control. B, Nuclear proteins were extracted, and then NF-κB p65 was detected by Western blot analysis. Effects of levosimendan and pimobendan on NF-κB nuclear translocation in cells were evaluated following 30-min incubation with LPS. Lamin B served as a nuclear marker. C, NF-κB promoter luciferase activity in RAW264.7 cells transfected with pGL4.32-NF-κB and pGL4.74-hRluc/TK. The transfected cells were stimulated with 1 μg/mL LPS for 6 hr. Levosimendan and pimobendan were given 30 min before LPS application. The results represent the ratio Firefly Luciferase/Renilla Luciferase. The data are presented as mean ± sem (n = 4). *p < 0.05 vs control.




LPS-stimulated RAW264.7 cells. This suggests that the inhibi-tory effect of levosimendan on overproduction of proinflam-matory and chemotactic cytokines cannot be solely attributed to alterations in the NF-κB pathway. Similarly, recent report has shown that levosimendan and its stereoisomer dextrosi-mendan did not affect the DNA binding of NF-κB in response to LPS in J774 macrophages (43).

LPS stimulation leads to phosphorylation of various intra-cellular signaling pathways, including MAPKs and Akt, and activation of these signaling pathways contributes to the regu-lation of expression of genes encoding proinflammatory and chemotactic cytokines in monocytes/macrophages (44–46). Some anti-inflammatory compounds that may be beneficial in septic animals have been reported to inhibit the MAPK path-way (47–49). On the other hand, our recent work has shown that the protective effects of the agents defined as a specific phosphodiesterase type III inhibitor or a direct adenylate cyclase activator on acute lung injury in CLP-induced septic mice are attributable to activation of the Akt pathway (22). However, levosimendan treatment was without effect on LPS-induced activation of MAPKs and Akt in macrophages, sug-gesting that the MAPK- and Akt-mediated signaling pathways are not a critical mechanism contributing to the ameliorating effect of levosimendan in septic mice.

In this study, we revealed that levosimendan is poten-tial inhibitor of HMGB1 release. HMGB1 is constitutively expressed in quiescent macrophage/monocytes, stored in the nucleus, and actively secreted from stimulated immune cells (34). In septic shock, HMGB1 could be secreted not only actively from activated immune cells, such as macrophages,

but also passively from injured or dying cells. Levosimendan prevented LPS-induced nuclear deprivation and extracel-lular release of HMGB1 in RAW264.7 cells. Levosimendan appears to inhibit LPS-induced HMGB1 release irrespec-tive of its transcriptional regulation. Indeed, HMGB1 mRNA levels were strongly decreased rather than increased in LPS-stimulated RAW264.7 macrophages, and its decrease was partially reversed by levosimendan treatment. We also found that neither LPS nor levosimendan altered HMGB1 mRNA levels in primary peritoneal macrophages isolated from mice (T. Imaizumi et al, unpublished observations, 2015). Outside the cells, HMGB1 can activate proinflammatory cytokine release from immune cells, including TNF and IL-1β (50). This may account for the possible mechanism(s) underlying the suppressive effect of levosimendan on overproduction of proinflammatory and chemotactic cytokines in in vivo and in vitro experiments. HMGB1 is considered as a critical regulator of sepsis severity. Blood HMGB1 concentrations are elevated in patients with sepsis (51). Patients with sepsis who succumbed to infection had higher serum HMGB1 levels than those who survived (52). We found that administration of levosimendan greatly suppressed the elevation in serum HMGB1 levels in CLP-induced septic mice. Our results thus indicate that HMGB1 may be one of the possible target mol-ecules for levosimendan in sepsis treatment. The secretion of HMGB1 from activated immune cells is likely to be a highly regulated process involving multiple steps (53, 54). A recent report has documented a key role of Rho kinase in HMGB1 secretion in sepsis (55). Indeed, we found that the specific Rho kinase inhibitor Y-27632 eliminated the release of HMGB1 in

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Figure 8. Effects of levosimendan (Levo) and pimobendan (Pimo) treatment on activation of mitogen-activated protein kinases (MAPKs) and Akt in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Levosimendan (30 μM) and pimobendan (100 μM) were given 30 min before application of 1 μg/mL LPS. Time course of changes in phosphorylation and total expression of extracellular signal-regulated protein kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), p38, and Akt after LPS challenge in the absence and presence of levosimendan or pimobendan are depicted. Results are representative of three independent experiments.


Wang et al


LPS-stimulated RAW264.7 cells. Interestingly, levosimendan suppressed the LPS-stimulated activation of RhoA, suggesting that levosimendan may have inhibited HMGB1 release by sup-pressing the Rho kinase signaling pathway. However, further study will be required to figure out the intricate mechanisms underlying the regulation of HMGB1 release in understanding the sepsis pathophysiology.

Pimobendan is another pyridazinone derivative calcium sensitizer and inhibited the up-regulation of proinflammatory and chemotactic cytokine expression in LPS-stimulated mac-rophages. Pimobendan behaved as the same way as levosimen-dan in terms of activation of the MAPK and NF-κB pathways, except that pimobendan, but not levosimendan, inhibited LPS-induced Akt activation in macrophages. However, whether the inhibition of Akt phosphorylation by pimobendan is respon-sible for its anti-inflammatory property appears questionable, as Akt is known to play a critical role in negatively regulating LPS-induced inflammation (32). In analogy with levosimen-dan, pimobendan resulted in a great inhibition of LPS-induced facilitation of HMGB1 release from the nucleus in macro-phages. We also found that continued administration of pimo-bendan strikingly decreased the elevations in serum HMGB1 levels in CLP mice. Our results represent evidence in favor of a class effect for the pyridazinone derivative calcium sensitizers with anti-inflammatory properties.

Levosimendan administration significantly improved the survival of mice after CLP. We showed that levosimendan mitigated cardiac dysfunction and hypotension, reduced lung injury, and inhibited the elevations in circulating levels of pro-inflammatory and chemotactic cytokines in CLP-induced sep-tic mice. These salutary effects may contribute to a reduction in septic mortality. Fluid therapy is one of the cornerstones of management of patients with sepsis (56). In this regard, our study design was fluid resuscitated after CLP. Thus, all animals used for survival study were given a subcutaneous injection of saline after the surgery because the impact of the levosimen-dan-induced increase in cardiac index on systemic hemody-namics might have been limited without fluid support.

One may argue that administration of levosimendan through an osmotic pump that was implanted into the peritoneal cavity following CLP does not accurately represent the clinical situation. To determine the potential practical relevance thereof, the investi-gation of levosimendan administration after sepsis induction may well deserve further study. Intriguingly, levosimendan was capable of inhibiting HMGB1 release and proinflammatory cytokine over-production in RAW264.7 cells even when it was added 30 minutes after LPS challenge, suggesting that postadministration of levosi-mendan may be effective in reducing endotoxic inflammation.

In conclusion, we demonstrate that levosimendan can mitigate hemodynamic failure, presumably arising from its

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Figure 9. Effects of levosimendan (Levo) and pimobendan (Pimo) treatment on high mobility group box (HMGB) 1 levels. A, HMGB1 messenger RNA (mRNA) in RAW264.7 cells. Lipopolysaccharide (LPS) (1 μM) was challenged for 24 hr following 30-min incubation with levosimendan (30 μM) or pimobendan (100 μM). HMGB1 expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. Values are mean ± sem of four separate experiments. ***p < 0.001 vs control. ###p < 0.001 vs LPS alone. B, The levels of HMGB1 released from RAW264.7 cells into the cell culture medium were measured by the enzyme-linked immunosorbent assay. Cells were stimulated with 1 μM LPS for 24 hr following 30-min incubation with 30 μM levosimendan. Values are mean ± sem of four separate experiments. ***p < 0.001 vs control. ###p < 0.001 vs LPS alone. C, Nuclear proteins were extracted, and then, HMGB1 was detected by Western blot. Effects of 30 μM levosimendan and 100 μM pimobendan on HMGB1 nuclear contents in RAW264.7 cells were evaluated following 24-hr incubation with LPS. Levosimendan and pimobendan were added 30 min before LPS. Lamin B served as a nuclear marker. D, Cells were given 30 μM levosimendan 30 min after LPS application. LPS was challenged for 24 hr, and then nuclear HMGB1 proteins was detected by Western blot. E, Effect of 50 μM Y-27632 on decreased HMGB1 nuclear contents following 24-hr incubation with LPS was assessed by Western blot. The final concentration of 0.5% of dimethyl sulfoxide (DMSO) used as a solvent did not affect the nuclear HMGB1 content. F, Ras homolog gene family, member A (RhoA) activity measurements. Cells were treated with or without 30 μM levosimendan. Cells were then further incubated with LPS for 5 min. Active guanine-5'-triphosphate (GTP)-bound RhoA was assayed by pull-down with GST-Rhotekin-Rho binding domain (RBD) coupled to colored glutathione-sepharose beads. G, Serum HMGB1 levels in 24-hr cecal ligation and puncture (CLP) mice. Levosimendan (0.5 μg/kg/min) or pimobendan (7.5 μg/kg/min) was continuously given through an osmotic pump that was implanted into the peritoneal cavity immediately following CLP. The data are presented as mean ± sem (n = 3–4 per group). *p < 0.05 vs control.




cardiac inotropy, but also confer substantial protection against cytokine-mediated inflammation in mice with CLP-induced sepsis. The critical step for levosimendan anti-inflammatory action can be partly explained by the current evidence that levosimendan is an effective inhibitor of HMGB1 release and its proinflammatory function. Thus, levosimendan with such anti-inflammatory profiles may potentially offer a therapeu-tic option as an inotrope in treatment of decompensated heart failure in patients with severe sepsis and septic shock.

ACKNOWLEDGMENTSWe thank Prof. Kuniaki Ishii (Department of Pharmacology, Yamagata University School of Medicine) for his helpful par-ticipation in the planning process of this study and Toshio Fujimori and Taiki Seki for their expert technical assistance. We are also grateful to Nippon Boehringer Ingelheim for sup-plying pimobendan.

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