Life Sciences xxx (2014) xxx–xxx

LFS-13992; No of Pages 7

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Significant reversal of cardiac upregulated endothelin-1 system in a ratmodel of sepsis by landiolol hydrochloride

Yoshimoto Seki, Subrina Jesmin, Nobutake Shimojo, Md. Majedul Islam, Md. Arifur Rahman,Tanzila Khatun, Hideaki Sakuramoto, Masami Oki, Aiko Sonobe, Junko Kamiyama, Keiichi Hagiya,Satoru Kawano, Taro Mizutani ⁎Department of Emergency and Critical Care Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan

⁎ Corresponding author at: Department of EmergenFaculty of Medicine, University of Tsukuba, Tsukuba, Ibar29 853 3210, 3081; fax: +81 29 853 5984.

E-mail address: mizutani@md.tsukuba.ac.jp (T. Mizuta

http://dx.doi.org/10.1016/j.lfs.2014.04.0050024-3205/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Seki Y, et al, Signhydrochloride, Life Sci (2014), http://dx.doi.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 3 November 2013Accepted 2 April 2014Available online xxxx

Keywords:HeartLandiolol hydrochlorideEndothelinSepsisRat model

Aims: Landiolol hydrochloride, an ultra-short-acting highly cardio-selective β-1 blocker, has become useful forvarious medical problems. Recent studies have demonstrated that co-treatment with landiolol protects againstacute lung injury and cardiac dysfunction in rats of lipopolysaccharide (LPS)-induced systemic inflammation,and was also associated with a significant reduction in serum levels of the inflammation mediator HMGB-1and histological lung damage. Endothelin (ET)-1, a potent vasoconstrictor, has been implicated in pathogenesisof sepsis and sepsis-inducedmultiple organ dysfunction syndrome. Here, we investigated whether landiolol hy-drochloride can play important roles in ameliorating LPS-induced alterations in cardiac ET system of septic rats.Main methods: Eight-week-old male Wistar rats were administered LPS only for 3 h and the rest were treatedwith LPS as well as with landiolol non-stop for 3 h.Key findings: At 3 h after LPS (only) administration, circulatory tumor necrosis factor (TNF)-α level, blood lactate

concentration and percentage of fractional shortening of heart were significantly increased. In addition, LPS in-duced a significant expression of various components of cardiac ET-1 system compared to control. Finally, treat-ment of LPS-administered rats with landiolol for 3 h normalized LPS-induced blood lactate levels and cardiacfunctional compensatory events, without altering levels of plasma TNF-α and ET-1. Most strikingly, landiololtreatment significantly normalized various components of cardiac ET-1 signaling system in septic rat.Significance: Taken together, these data led us to conclude that landiololmay be cardio-protective in septic rats bynormalizing the expression of cardiac vasoactive peptide such as ET, without altering the circulatory levels of in-flammatory cytokines.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Sepsis is associatedwith tissuehypoperfusion andmetabolic impair-ment, whichmay contribute to the development of the associated mul-tiple organ failure (Singh and Evans, 2006). As an important organsystem that is commonly vulnerable to sepsis or septic shock, thecardiovascular system and its dysfunction have been studied in bothclinical and basic research for a long time. When sepsis-induced cardio-vascular dysfunction was first described by Waisbren in 1951, it wasrecognized as a hyperdynamic state with full bounding pulses, flushing,fever, oliguria and hypotension (Waisbren, 1951). More recently, echo-cardiographic studies demonstrated impaired left ventricular systolic

cy and Critical Care Medicine,aki, 305-8575 Japan. Tel.: +81

ni).

ificant reversal of cardiac uporg/10.1016/j.lfs.2014.04.005

and diastolic dysfunction in septic patients (Poelaert et al., 1997). Cardi-ac dysfunction in sepsis is characterized by decreased contractility, im-paired ventricular response to fluid therapy, and in some patients,ventricular dilatation (Zanotti-Cavazzoni and Hollenberg, 2009). Cur-rent data support a complex underlying physiopathology of sepsis-induced myocardial dysfunction, with a host of potential pathwaysleading to myocardial depression (Zanotti-Cavazzoni and Hollenberg,2009). Cytokines, such as tumor necrosis factor (TNF)-α, interleukin(IL)-1, and lysozyme C, have direct inhibitory actions on cardiomyocytecontractility (Horton et al., 2000; Kumar et al., 2007; Mink et al., 2005;Zanotti-Cavazzoni and Hollenberg, 2009). Endothelin (ET)-1 is alsosaid to be a major factor (Zanotti-Cavazzoni and Hollenberg, 2009).ET-1 is the most potent vasoconstrictor studied so far (Yanagisawaet al., 1988), and its (ET-1) upregulation has been demonstrated within6 h of inducing sepsis/septic shock by lipopolysaccharide (LPS) in ani-mal models (Shindo et al., 1998). Overexpression of ET-1 contributesto an increase of inflammatory cytokines, especially TNF-α, IL-1, andIL-6, and an inflammatory cardiomyopathy that results in cardiac

regulated endothelin-1 system in a rat model of sepsis by landiolol

2 Y. Seki et al. / Life Sciences xxx (2014) xxx–xxx

dysfunction (Yang et al., 2004). Konrad et al. reported that tazosentan, adual endothelin type A (ET-A) and endothelin type B (ET-B) receptorantagonist improves cardiac index, stroke volume index, and left ven-tricular stroke work index in sepsis mouse model (Konrad et al.,2004). These findings support the involvement of ET-1 in myocardialdysfunction in sepsis. However, further investigations are needed inorder to develop effective therapeutic strategies.

Landiolol, an ultra-short-acting and highly cardio-selective β-1blocker, has become useful for various medical problems. The firststudy of β-blocker therapy for septic shock was published in 1969(Berk et al., 1969). More recently, studies have demonstrated that co-treatment with landiolol protects against acute lung injury and cardiacdysfunction in a rat model of LPS-induced systemic inflammation,which was also associated with a significant reduction in serum levelsof the inflammation mediator HMGB-1 and histological lung damage(Hagiwara et al., 2009). However, the effects of landiolol on the LPS-altered ET signaling system in the heart of rat are yet to be demonstrated.

In the present study, we investigated whether landiolol hydrochlo-ride can play an important role in ameliorating the LPS-induced alter-ation in cardiac ET signaling system in a rat model of sepsis.

Materials and methods

Animal preparation

Male Wistar rats (200–250 g, 8 weeks old) were used in all experi-ments. Sepsis was induced by the intraperitoneal (IP) administrationof bacterial LPS from Escherichia coli 055:B5 (15 mg/kg), dissolved insterile saline. The LPS dose used in the present study has been used be-fore to generate animal models of sepsis in our several previous studies(Jesmin et al., 2004, 2006, 2007, 2009a,b, 2011a,b; Shimojo et al., 2006a;Yamaguchi et al., 2006; Zaedi et al., 2006). The dose of LPS used in thecurrent investigation as well as that in the previous studies producethe clinical condition that resembles sepsis in context of circulatory aswell as tissue and organ dependent alteration ranging frommorpholog-ical to gene expression changes (Jesmin et al., 2004, 2006, 2007, 2009a,b,2011a,b; Shimojo et al., 2006a; Yamaguchi et al., 2006; Zaedi et al.,2006). The current LPS dose has been shown to demonstrate minimalmorphological injuries in important organs prone to develop dysfunc-tion during sepsis (Jesmin et al., 2004, 2006, 2007, 2009a,b, 2011a,b;Shimojo et al., 2006a; Yamaguchi et al., 2006; Zaedi et al., 2006).The control group received an equal volume of vehicle (sterile saline;2 ml/body) intraperitoneally,without LPS. In every studyour group con-ducts, we always confirm the induction and generation of sepsis modelby performing a detailed assessment of inflammatory cytokines, notablylevels of circulatory and tissue TNF-α, iNOS, IL-6, blood gas analysis,morphological and functional evaluation of organs likely prone to sepsis(Jesmin et al., 2004, 2006, 2007, 2009a,b, 2011a,b; Shimojo et al., 2006a;Yamaguchi et al., 2006; Zaedi et al., 2006). The different groups of ani-mals (n = 25) in the present study were killed by Nembutal (sodiumpentobarbital, IP, 80 mg/kg body weight) at 3 h after LPS or vehicleonly. The blood samples were collected from a polyproprene tube cath-eter inserted into the left carotid artery for blood gas analysis, and hearttissues were harvested gently, frozen immediately in liquid nitrogen,and stored at −80 °C. All animals received care and all experimentalprocedures were approved by the Animal Care and Use Committee ofUniversity of Tsukuba. It should be noted that LPS (15 mg/kg, intrave-nous) was dissolved in normal saline administered intravenously attime 0 to different groups of rats, and then the rats were killed after 3h. However, for the LPS+ landiolol hydrochloride group, 15 min beforeLPS administration, landiolol hydrochloride was administered intrave-nously continuously for 3 h (100 μg/kg/min).

Our preliminary time course study conducted at various time points(0 h, 1 h, 3 h, 6 h, 10 h, 16 h, 24 h, n = 13 for each group) showed thatLPS induced high levels of plasma lactate, elevated heart rate (HR) andincreased percent of fractional shortening (% FS), i.e., a hyperdynamic

Please cite this article as: Seki Y, et al, Significant reversal of cardiac uphydrochloride, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.04.005

state during the early hours of sepsis. These elevated end-points weregreatly normalized significantly by landiolol as early as 3 h comparedto the LPS only administered rats. This reversal of the hyperdynamicstate during septic shock by landiolol resulted inmaintaining low levelsof essential cardiac output, systemic peripheral circulation and arterialoxygenation at all time points of sepsis, and consequently leading toan improved survival rate. Based on these facts, we chose the 3 h timepoint to investigate in details, the effects of landiolol on septic ratheart. On the other hand, the time duration of sepsis induction (3 h)in the present study may have little to do with the pathology in clinicalsepsis response which may have a time course of days to weeks. How-ever, the present study aimed to focus on the acute response phase ofsepsis especially in context of hemodynamic alteration in experimentalanimal model.

Measurements of hemodynamic parameters

The ratswere anesthetizedwith isoflurane inhalation (1.5%, 1 L/min)and a microtip pressure transducer catheter (SPC-320, Millar Instru-ments, Houston, TX, USA) was inserted into the left carotid artery, as de-scribed in previous study. Then arterial blood pressure and heart ratewere monitored with a pressure transducer (model SCK-590, Gould,Ohio, USA) and recorded with the use of a polygraph system (amplifier,AP-601G, Nihon Kohden, Tokyo, Japan; tachometer, AT-601G, NihonKohden; and thermal-pen recorder, WT-687G, Nihon Kohden).

Echocardiography

Echocardiographywasperformedusing a Vevo 2100high-frequencyultrasound system (Visual Sonics Inc, Ontario, Canada), which includesan integrated rail system for consistent positioning of the ultrasoundprobe (Yang et al., 2013). The hair from the chest was removed withan electrical clipper and a hair removal gel prior to the examination.The animals were placed on a heating pad and connected to an electro-cardiogram (ECG) while rectal temperature was monitored to maintainbody temperature 38 ± 0.1 °C. A 35 MHz linear transducer (VisualSonics, RMV 707, Inc, Ontario, Canada) was used for imaging. An opti-mal parasternal long axis (LAX) cine loop (i.e. visualization of both themitral and aortic valves, and maximum distance between the aorticvalve and the cardiac apex) of N1000 frames/s was acquired using theECG-gated kilohertz visualization technique. The probe was then rotat-ed 90° and positioned 6mmbelow themitral annulus, i.e. at the level ofthe papillary muscles. Three parasternal short axis (SAX) M-mode se-quences were stored. Fractional shortening (FS) was calculated in theM-mode image as FS = (EDD − ESD)/EDD, where EDD and ESD areend-diastolic and end-systolic diameters, respectively.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELIZA), a sensitive techniquefor determining circulatory and tissue protein concentration, was usedto determine levels of IL-6 and TNF-α in serum and ET-1 in plasmaand heart tissue extracts (R&D Systems, Minneapolis, MN) accordingto the manufacturer’s protocol. For ET-1 measurement, a 4.5 h solidphase ELIZA was used, and contained synthetic ET-1 and antibodiesraised against synthetic ET-1. This immunoassay has been shown to ac-curately quantitate synthetic and naturally occurring ET-1. A monoclo-nal antibody specific for ET-1 was pre-coated onto a microplate.Standards and samples were pipetted into the wells and if present, ET-1 antigen was bound by the immobilized antibody. After washingaway any unbound substances, an enzyme-linkedmonoclonal antibodyspecific to ET-1was added to thewells. Following awash to remove anyunbound antibody-enzyme reagent, a substrate solution was added tothe wells and a color developed in proportion to the amount of ET-1bound in the initial step. The color development was then stoppedand its intensity measured. The ET-1 concentration of each sample

regulated endothelin-1 system in a rat model of sepsis by landiolol

Table 1Hemodynamic, echocardiogram, and blood gas analysis parameters in experimentalanimals.

Control LPS LPS + landiolol

Systolic BP, mmHg 123 ± 3.5 102 ± 4.4** 98 ± 6.6**Diastolic BP, mmHg 83 ± 4.2 75 ± 3.1** 73 ± 5.3**Heart Rate, bpm 459 ± 16 502 ± 16** 440 ± 6##

Fractional Shortening, % 40.94 ± 1.38 45.07 ± 1.18* 41.16 ± 0.99#

pH 7.43 ± 0.01 7.42 ± 0.02 7.40 ± 0.02PaO2, torr 102.5 ± 2.7 90.4 ± 11.7* 100.3 ± 5.7PaCO2, torr 38.7 ± 2.0 34.1 ± 1.8* 32.2 ± 3.2*Base Excess, mmol/l 1.0 ± 0.7 −1.8 ± 1.0 −5.1 ± 1.2*Lactate, mmol/l 0.9 ± 0.2 3.0 ± 0.5* 1.6 ± 1.2*#

HCO3−, mmol/l 25.4 ± 0.8 22.2 ± 0.9* 20.1 ± 1.4*

Data are mean ± SE; *p b 0.05 vs. control; **p b 0.01 vs. control; #p b 0.05 vs. LPS;##p b 0.05 vs. LPS.Abbreviations: BP, blood pressure; LPS, lipopolysaccharide.

3Y. Seki et al. / Life Sciences xxx (2014) xxx–xxx

was calculatedwith a standard curve constructed by plotting the absor-bance of each standard solution.

Western blotting

Ice-cold heart tissuesweremincedwith scissors, homogenized, cen-trifuged and then the concentration of the protein (supernatant) wasdetermined using the bicinchoninic acid protein assay (Pierce Biotech-nology). Samples were boiled in reducing SDS sample buffer for 5 min,loaded onto an SDS-PAGE (4–15% polyacrylamide) gel, subjected toelectrophoresis, and electrophoretically transferred to polyvinylidenedifluoride filter membrane. To reduce non-specific binding, the mem-brane was blocked for 2 h at room temperature with 5% non-fat milkin PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4)containing 0.1% Tween 20, incubated overnight at 4 °C with primaryantibodies in PBS-Tween buffer, washed three times with PBS-Tweenbuffer, and then themembranewas incubatedwith a suitable secondaryantibody coupled to horseradish peroxidase for 60min at room temper-ature. The blots were thenwashed three times in PBS-Tween buffer andsubsequently visualized with an enhanced chemiluminescence detec-tion system (Amersham) and exposed to an X-ray film (Fuji PhotoFilm). The intensity of total protein bands per lane were evaluated bydensitometry. Negligible loading/transfer variation was noted betweensamples. Moreover, β-actin was used as loading control. Commerciallyavailable and well-characterized antibodies were used in the presentstudy, including ET-A receptor antibody (Alomone Labs, Jerusalem,Israel), ET-B receptor antibody (Alomone Labs, Jerusalem, Israel) andendothelin-converting enzyme-1 (ECE-1, Santa Cruz Biotechnology,Dallas, TX). The enzyme that transforms Big ET-1 into ET-1 is designatedas ECE-1 and acts in a highly specificmanner (Xu et al., 1994). ECE-1 is akey enzyme in the biosynthesis of ET-1 because the biological activitiesof Big ET-1 are negligible (Xu et al., 1994).

RNA preparation and real-time quantitative polymerase chain reaction

Total RNA from heart tissue was isolated using RNeasy (Qiagen,Tokyo, Japan). After isolation, DNase I treatment, and quantification,RNA was reverse transcribed to cDNA by Omniscript RT using a first-strand cDNA synthesis kit (Qiagen). The reaction was performed at37 °C for 60 min.

The mRNA expression levels of target genes were analyzed by real-time quantitative PCR with TaqMan probe using an ABI Prism 7700 se-quence detector (Perkin-Elmer Applied Biosystems, Foster, CA). Thegene-specific primers and TaqMan probeswere synthesized fromPrimerExpress version 1.5 software (Perkin-Elmer), according to the publishedcDNA sequences for each gene, as previously described (Shimojo et al.,2006b, 2007).

The expression of GAPDHmRNAwasused as an internal control. ThePCR mixture (25 μl total volume) consisted of forward and reverseprimers for each gene (Perkin-Elmer) at 450 nM each, FAM-labeledprimer probes (Perkin-Elmer) at 200 nM, and TaqMan Universal PCRMaster Mix (Perkin-Elmer). Each PCR amplification was performed intriplicate as follows: 1 cycle at 95 °C for 10 min and 40 cycles at 94 °Cfor 15 s and 60 °C for 1 min. The quantitative values of target mRNAswere normalized by GAPDHmRNA, because GAPDHmRNA expressionswere more stable among all the samples than other internal controls,such as β-actin and 18S ribosomal RNA. Primers and probes are as fol-lows: ET-1 forward: 5′-TCTACTTCTGCCACCTGGACAT-3′, ET-1 reverse:5′-GAAGGGCTTCCTAGTCCATACG-3′, and ET-1 probe: 5′-CATCTGGGTCAACACTCC-3′; ET-A forward: 5′-GAATCTCTGCGCTCTCAGTGT-3′, ET-Areverse: 5′-GAGACAATTTCAATGGCGGTAATCA-3′, and ET-A probe: 5′-CAGGAAGCCACTGCTCT-3′; ET-B forward: 5′-GCTGGTGCCCTTCATACAGA-3′, ET-B reverse: 5′-CTTAGAGCACATAGACTCAACACTGT-3′, ET-Bprobe: 5′-ATCCCCACAGAAGCCT-3′; ECE-1 forward: 5′-TCAGACAAGTCTCCACACTCATCA-3′, ECE1 reverse: 5′-CCAGGTTCCACATCATGTAGTTGTT-3′, ECE1 probe: 5′-ACAGCACCGACAAATG-3′; GAPDH forward: 5′-

Please cite this article as: Seki Y, et al, Significant reversal of cardiac uphydrochloride, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.04.005

GTGCCAAAAGGGTCATCATCTC-3′, reverse: 5′-GGTTCACACCCATCACAAACATG-3′, and probe: 5′-TTCCGCTGATGCCCC-3′.

Statistical analysis

The results were expressed asmean±SE, and themeanswere com-pared by a one-way factorial analysis of variance, followed by Scheffé’stest for multiple comparisons. Differences were considered significantat p b 0.05.

Results

Both the systolic anddiastolic blood pressure levelswere significant-ly lower at 3 h after LPS administration and were unaffected with thetreatment of landiolol for 3 h (Table 1). Heart rate was significantly in-creased in LPS group (p b 0.05) compared to control group and signifi-cantly decreased in LPS-administered rats treated with landiolol(p b 0.05) (Table 1). Percent (%) FS measured with echocardiographwas also significantly hyperkinetic in LPS-administered group(p b 0.05) compared to control (Table 1). However, hyperdynamicstate induced by LPS administration was significantly normalized inLPS + landiolol group (p b 0.05 vs LPS) (Table 1). Arterial PaO2 wasfound to be significantly reduced in LPS-administered rats and landiololhad no significant effect on arterial PaO2 (Table 1). Blood lactate concen-trationswere increased dramatically after LPSwas given and partly nor-malized with the treatment of landiolol (Table 1).

Serum levels of TNF-α and IL-6, which are inflammatory cytokines,determined by ELIZA were significantly increased after LPS administra-tion. However, landiolol treatment did not change levels of these mole-cules (Fig. 1A and B) in septic rats. Plasma ET-1 level also significantlyincreased in LPS and LPS + landiolol groups compared with control(Fig. 1C). Landiolol had no effect on elevated ET-1 levels in septic rats,as with the other two pro-inflammatory factors.

Prepro-ET-1 mRNA levels were elevated in heart tissue after LPS ad-ministration, compared to the control group and landiolol treatmentameliorated the elevation of prepro-ET-1 mRNA significantly (Fig. 2A).ET-A receptor mRNA levels in heart tissue also increased markedlyafter LPS administration, and landiolol had no significant effect on its(ET-A receptor) expression (Fig. 2B). ET-B receptor and ECE-1 mRNAexpressions were also significantly increased in LPS group and landiololtreatment significantly normalized their expression (Fig. 2C and D).

The mRNA expression data of various components of ET systemcorresponded with expression of their respective proteins, as demon-strated by ELIZA and immunoblotting experiments (Fig. 3). CardiacET-1 levels were significantly higher in LPS-administered group com-pared to control group and was greatly normalized by landiolol admin-istered for 3 h (Fig. 3A). The protein expressions of both ET receptorswere significantly higher in heart tissues after LPS administration at 3h compared to the respective protein expression in the control group

regulated endothelin-1 system in a rat model of sepsis by landiolol

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Fig. 1. Serum TNF-α (A); serum IL-6 (B) and plasma ET-1 (C) levels from the control, 3 hLPS-administered rats, and 3 h landiolol-treated LPS-administered rats. These data weregenerated by ELIZA, as described above. Values are mean ± SE (n = 12). *p b 0.05 vs.control.

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Fig. 2. Gene expression levels of prepro-ET-1 (A), ET-A (B), ET-B (C) receptors, and ECE-1

4 Y. Seki et al. / Life Sciences xxx (2014) xxx–xxx

(Fig. 3B and C). Three hours of treatment with landiolol significantlynormalized the upregulated expression of ET-B receptor in the heartof LPS-administered septic rat. However, it (landiolol) failed to normal-ize the upregulated ET-A receptor expression significantly (Fig. 3B andC). Cardiac ECE-1 protein expressions were also significantly increasedafter LPS administration at 3 h compared to the control group andlandiolol treatment significantly normalized ECE-1 upregulation inheart tissue of septic rats (Fig. 3D).

(D) in heart tissues of the control, 3 h LPS-administered rats, and landiolol treated 3 h LPS-administered rats. Gene expression levels were analyzed by real-time quantitative PCR.GAPDHwas used as internal control. In each of the experiments, the control was normal-ized as 100%. Values are mean ± SE (n = 12). *p b 0.05 vs. control, #p b 0.05 vs. 3 h LPS-administered rats.

Discussion

In the present study, we have demonstrated that (1) LPS significant-ly upregulates expression of various protein and mRNA components ofET-1 signaling system in the cardiac tissues compared to levels of thecontrol; (2) treatment of LPS-administered rats with landiolol forthree continuous hours normalized elevated levels of blood lactateand cardiac functional compensatory events, but without altering levels

Please cite this article as: Seki Y, et al, Significant reversal of cardiac uphydrochloride, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.04.005

of plasma TNF-α and ET-1. Most strikingly, landiolol treatment signifi-cantly normalized the various components of the elevated ET-1 signal-ing system in the heart of the septic rat.

regulated endothelin-1 system in a rat model of sepsis by landiolol

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Fig. 3. Protein expression levels in cardiac tissue of ET-1 (A) peptide, ET-A (B), ET-B (C) receptors, and ECE-1 (D) from the control, 3 h LPS-administered rats, and 3 h landiolol-treated LPS-administered rats. (A) Datawere generated by ELIZA, as described above. (B–D) Immunoblot analysis of ET-A (B), ET-B (C) receptors, and ECE-1 (D) (typicalWestern blots and intensity ofthe bands). In each experiment, the band obtained with control is normalized as 1.0. We used β-actin as internal control. Values are mean ± SE (n= 16). *p b 0.05 vs. control, #p b 0.05vs. 3 h LPS-administered rats.

5Y. Seki et al. / Life Sciences xxx (2014) xxx–xxx

Organ failure occurs in 33.6% of septic shock patients, which mayreach as much as 70% in cases that have severe hypotension (Martinet al., 2003). Cardiac dysfunction in sepsis is characterized by ventriculardilatation, reduction in ejection fraction and reduced contractility(Flynn et al., 2010). Initially, cardiac dysfunction was considered tooccur only during the “hypodynamic” phase of shock (Flynn et al.,2010). However, we now know that it occurs very early in sepsis, in-cluding the “hyperdynamic” phase of septic shock (Flynn et al., 2010).Accordingly, at 3 h after LPS administration in the present study, wefound hyperdynamic state in the current rat model of sepsis. Patholog-ically over-enhanced cardiac function (hyperdynamic state in sepsis)compensating systemic circulation in the early phase of sepsis may failto the collapse of cardiac function and systemic circulation in latephase. The underlying mechanisms of these cardiac events are notwell understood, but involve circulating factors, such as neutrophiladhesion to the endothelium, TNF-α, IL-1, production of reactive freeradicals and oxidants, activation of toll-like receptors, cardiomyocyteapoptosis and endothelial dysfunction (Lorigados et al., 2010; Zouet al., 2010).

ET-1, a potent vasoconstrictor, has been implicated in the pathogen-esis of sepsis and sepsis-induced multiple organ dysfunction syndrome(Furian et al., 2012; Piechota et al., 2007). Previous studies have sug-gested that ET-1 is a direct inhibitor of myocardial contractility(Wanecek et al., 1999, 2001). A number ofmechanisms regulated by va-soactive mediators, such as ET-1, nitric oxide and mitogen-activatedprotein kinase pathways, have been proposed to affect myocardial

Please cite this article as: Seki Y, et al, Significant reversal of cardiac uphydrochloride, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.04.005

performance during sepsis. Previous studies demonstrated that hyper-dynamic state in sepsis produced an elevated concentration of ET-1 upto 12 h post-sepsis, which returned to basal levels at 24 h (Sharmaet al., 1997). In our present study, we also found elevated ET-1 levelsin the heart tissues at 3 h after LPS administration during thehyperdynamic state. This upregulation was accompanied by an in-creased expression of both ET receptors, aswell as the enhanced expres-sion of ECE in septic heart. In a very recent study, it was demonstratedthat septic acute heart failure is attributed to downregulation ofFKBP12.6 and SERCA2a, which is related to an activated ET system (Heet al., 2007). An ET receptor antagonism of CPU0213 significantly im-proves cardiac performance by blocking both ET-A and ET-B receptors(He et al., 2007). Thus a number of studies have clearly demonstratedthe involvement of ET system in myocardial pathogenesis in sepsis.

Landiolol, an ultra-short-acting (a half-life of 4 min) and highlycardio-selectiveβ-1 blocker, has becomeuseful in treating variousmed-ical problems, such as the management of acute rapid heart rate, pre-vention of atrial fibrillation after cardiac surgery (Iguchi et al., 1992;Momomura, 2013; Sakaguchi et al., 2012). The drug has already beenapproved as an emergency treatment of supraventricular tachyarrhyth-mia in patients in Japan. However, landiolol has been reported to have alesser effect on blood pressure compared to esmolol (Yamazaki et al.,2005). Recent studies have demonstrated that co-treatment withlandiolol protects against acute lung injury and cardiac dysfunction ina ratmodel of LPS-induced systemic inflammation,whichwas also asso-ciated with a significant reduction in serum levels of the inflammation

regulated endothelin-1 system in a rat model of sepsis by landiolol

6 Y. Seki et al. / Life Sciences xxx (2014) xxx–xxx

mediatorHMGB-1 and histological lung damage (Hagiwara et al., 2009).However, whether landiolol has any effect on ET system in sepsis is yetto be demonstrated.

Treatment of LPS-administered rats with landiolol for 3 h normal-ized the elevated levels of blood lactate and cardiac functional compen-satory events in septic rats. In the present study, we were unable tomeasure preload, such as central venous or left ventricular end-diastolic pressures. However, we transfused the same volume of salineto each experimental rat group. Moreover, cardiac output calculatedby echocardiographic data showed no significant change in all thethree experimental groups. Moreover, lactate concentration in bloodwas significantly decreased when landiolol was administered to septicrats. These data collectively led us to speculate that landiolol couldcounteract LPS-induced hyperdynamic state in the heart and maintainperipheral circulation. Further, this highly cardio-selective β-1 blockerdid not also induce vasodilation in peripheral vessels, thus preventingdevelopment of extreme hypotension during septic shock. Althoughthe present study reveals a significant decrease in blood pressure afterLPS administration, no abrupt drop in blood pressure was observedafter the treatment of landiolol in septic rat. Thus, the change in frac-tional shortening with LPS (hyperdynamic state in sepsis) can be ex-plained by the decrease in afterload as observed in the present study;however, the decrease (normalization of fractional shortening) withlandiolol occurs without a significant change in afterload compared toLPS alone in current experimental design. There might be someunknown/unexplored effects of landiolol on hemodynamics in sepsisbesides the known conventional pharmacological actions of landiololwhich need to be explored and addressed in future studies.

One of themost important findings of the present study is the rever-sal of elevated levels of cardiac ET-1 in septic rats by landiolol. Increasedcardiac ET production during sepsis contributes to depressed cardiacperformance. We show here that landiolol can counteract this cardiacimpairment because of its inhibitory effect on the expression of ET-1in the heart of septic rats. This implies that landiolol may be cardio-protective in septic rats by normalizing the expression of cardiac vaso-active peptide like ET without altering the circulatory level of ET-1. Infact, β1-adrenoceptors are selectively expressed in myocardium(Wallukat, 2002). Therefore, landiolol, a selective β1-adrenoceptorblocker, was able to reduce the upregulated ET-1 in heart tissue greatlyin sepsis without exerting a significant reversal effect on plasma ET-1concentration. For the moment, we have no explanation underlyingthe differential pattern of landiolol effects on circulating level of ET-1(not affected by landiolol) and myocardial ET-1 (reduced by landiolol)as observed in the present study.Moreover, landiolol exerted significanteffect on the normalization of ET-B receptors in heart of septic rat with-out altering levels of ET-A receptors. Finally, we state that the normaliz-ing effect of landiolol on the elevated levels of cardiac ET signalingsystem did not depend on changes in TNF-α. It is now evident fromthe present findings and the previous studies (Hagiwara et al., 2009)that landiolol exerts differential effects on levels of circulatory inflam-matory cytokines in septic subject.

For now, we do not have an adequate explanation for the observedreversal in the expression of the elevated cardiac ET signaling systemby landiolol in the sepsis animal models. Some reports showed thatcarvedilol, which is both an α and β adrenergic receptor blocker,suppressed pathologically overexpressed myocardial ET-1, consistentwith the current findings (Massart et al., 1999; Zhao et al., 2008).Thus, collectively, findings from the present and previous studies indi-cate that adrenergic receptor blockers, such as carvedilol and landiolol,can significantly normalize elevated levels of cardiac ET-1 in variouspathological conditions. However the exact mechanisms underlyingsuch reversal effects on the elevated levels ofmyocardial ET-1 by adren-ergic receptors blockers remain to be explored. Further, we speculatethat landiolol might downregulate prepro-ET-1 in the heart of LPS-induced septic rats via normalizing HIF-1α expression. In our unpub-lished observation we found that HIF-1α expression is significantly

Please cite this article as: Seki Y, et al, Significant reversal of cardiac uphydrochloride, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.04.005

upregulated in septic heart compared to control rat heart tissue. Further,a recent study demonstrated that HIF-1α expression is upregulated inthe heart of a septic rat, 5 h post-LPS administration (Bateman et al.,2007). Indeed, HIF-1 is one of transcriptional factors for ET-1 incardiomyocytes (Kakinuma et al., 2002). Thus, taken together, we con-clude that sepsis-induced adrenergic “storm”may systemically upregu-late HIF-1α and ET-1 expression in heart tissue and that the subsequentenhanced expressions can be ameliorated by β adrenergic blockade.This conclusion is further validated by findings from recent studiesthat have shown that activation of the PI3K and p42/p44 MAPK path-ways, one of the downstream components of adrenergic receptors, in-duce synthesis of HIF-1α via the action of mTOR (Laughner et al.,2001; Lee et al., 2006). On the other hand, Zhao et al. reported that car-vedilol reduces myocardial no-reflow by decreasing ET-1 by activatingthe ATP-sensitive K+ channel (Zhao et al., 2008). Thus, several path-ways/mechanisms underlying the suppressive effects of landiolol onmyocardial ET-1 levels in septic rats may be involved and should beclarified in future studies.

The present study is the first to demonstrate the beneficial effects oflandiolol on the heart of a septic rat, by assessing broad alterations rang-ing from molecular to pathophysiological end-points such as hemody-namics and expression of pro-inflammatory factors. β-Blockers areconventionally considered to disturb/interrupt the physiological com-pensatory system in septic heart responsible for maintaining systemiccirculation. Specifically, we demonstrated the potential of a β-blockerin regulating the hemodynamic compensatory state during septicshock. Further, the currentfindings on use ofβ-blocker can be expandedto other sepsis-induced cardiac complications. In the present study, be-cause of the elucidation of the mechanism of landiolol effect to septicrats, we administered landiolol prior (pre-treatment) to the LPS admin-istration in experimental animals. Some clinical studies have shownthat hypertensive patients treated with chronic β-blockers comparedto patients lacking β-blockers had good prognosis after failing in septicshock (Macchia et al., 2012). However, the effects of landiolol throughpre-treatment approach to septic models demonstrated in the currentinvestigation may have limitation in extrapolating to relevant clinicalsituations. Future studies with various treatment designs of landiololshould be investigated using the septic models. The present findingswould at leastmake a base onwhich to explore and dissect the potentialclinical implication of β-blocker, such as landiolol for septic subjects. Inaddition, in depth studies aimed at teasing out the underlying mecha-nisms of the observed effects of landiolol in sepsis reported here shouldbe conducted in future studies using genetically engineered animalmodels. The role of blood pressure on the effects of landiolol from thehemodynamics to the molecular level in sepsis should be also investi-gated in depth in future studies.

Conclusion

Taken together, these data led us to conclude that landiolol may becardio-protective in septic rats by normalizing the expression of cardiacvasoactive peptide such as ET without altering the circulatory levels ofpotential inflammatory cytokine, like TNF-α. These findings may pro-vide new windows of opportunities for future explorations on the useof landiolol in sepsis-induced multiple organ dysfunction syndrome.

Conflict of interest statement

None.

Acknowledgements

This work was supported in part by a Grant-in-Aid for ScientificResearch B and C from the Ministry of Education, Culture, Sports,Science and Technology of Japan (22390334, 23592025, 23406037,

regulated endothelin-1 system in a rat model of sepsis by landiolol

7Y. Seki et al. / Life Sciences xxx (2014) xxx–xxx

23406016, 23406029, 24406026, 25462812 and 25305034) and JapanSociety for the Promotion of Science.

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