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Improved Myocardial Function With Supplement of Levosimendan to Celsior Solution

Zhou, Hai-yan MD*; Zhang, Li-na BS*; Zheng, Ming-zhi MD; Wang, Lin-lin PhD; Chen, Ying-ying PhD; Shen, Yue-Liang MD

Journal of Cardiovascular Pharmacology: September 2014 - Volume 64 - Issue 3 - p 256–265
doi: 10.1097/FJC.0000000000000115
Original Article
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Abstract: Levosimendan is a calcium-sensitizing agent shown to prevent myocardical contractile depression in various heart diseases. In this study, we investigated the effect of levosimendan on cardiac dysfunction and apoptosis in hypothermic preservation rat hearts. Isolated rat hearts were preserved in Celsior solution with or without levosimendan. The left ventricular developed pressure (LVDP) recovery rate of isolated rat heart significantly decreased, and the apoptosis index increased after 9 hours of hypothermic preservation. Supplement Celsior solution with levosimendan (10−7 and 10−6 mole/L) enhanced the LVDP recovery rate and reduced apoptosis. Levosimendan inhibited the hypothermic preservation-induced calpain activation and cleavage of Bid. Levosimendam induced increased myocardial inducible nitric oxide synthase but not endothelial nitric oxide synthase expression. A selective inducible nitric oxide synthase inhibitor 1400W, and a mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-hydroxydecanoate but not a sarcolemmal KATP channel blocker HMR-1098 prevented improvement effect of levosimendam on LVDP recovery rate, abolished the inhibitory effect of levosimendan on hypothermic preservation-induced activation of calpain, cleavage of Bid, and apoptosis. These data suggested that Celsior solution supplement with levosimendan improved cardiac function recovery and reduced myocyte apoptosis in hypothermic preservation rat hearts.

*Department of Anesthesiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China;

Department of Pharmacology, Zhejiang Medical College, Hangzhou, China; and

Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, China.

Reprints: Yue-Liang Shen, MD, Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou 310058, China (e-mail: shenyueliang@zju.edu.cn).

Supported by the National Natural Science Foundation of China (No. 81070201 and 81270178).

The authors report no conflicts of interest.

The authors had full access to all data in the study and final responsibility for the decision to submit for publication.

This is an open access article distributed under the terms of the Creative Commons Attribution-Noncommercial No Derivative 3.0 License, which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially.

Received December 21, 2013

Accepted April 13, 2014

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INTRODUCTION

Heart transplantation has become the standard therapy for end-stage heart failure and severe coronary artery diseases.1,2 Major clinical and laboratory research in the field of heart transplantation have been focused on expanding the donor pool, refining the use of immunosuppression, and monitoring the effects of therapy.1 Because serious ischemia–reperfusion (I/R) injury during heart transplantation is a major cause of perioperative morbidity and mortality, successful organ preservation becomes more imperative to reduce the reperfusion injury in clinical heart transplantation practice.

Levosimendan is a calcium-sensitizing agent that exerts its hemodynamic properties by positive inotropy, vasodilation, and cardioprotection. Rump et al3 first reported anti-ischemic effects of levosimendan in isolated rabbit hearts. Levosimendan decreases mortality in acute episodes of decompensated heart failure by increasing myocardial contractility, decreasing preload or afterload.4 Levosimendan also improves cardiac function and survival in patients with chronic advanced heart failure.5 In cardiac I/R animal models, levosimendan decreases infarct size by modulation of programmed forms of cell death.6,7 Oral treatment with levosimendan improves cardiac functions and prevent cardiac remodeling after myocardial infarction in a rodent model of type 2 diabetes.8

Apart from being used as a protective agent against I/R injury, effects of levosimendan have also been assessed in cardioplegic solutions and during cardiac arrest.9,10 However, evidence of beneficial effects of levosimendan-supplemented Celsior solution in hypothermic preservation-induced cardiac injury is limitted. In this study, we aimed to determine the effect of levosimendan on cardiac dysfunction and apoptosis in hypothermic preservation rat hearts.

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METHODS

Animals

Male Sprague–Dawley rats (220–240 g) were purchased from the Experimental Animal Center of Zhejiang University and cared for in compliance with the Guide for the Care and Use of Laboratory Animals published by National Institutes of Health.

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Reagents

Levosimendan, 5-hydroxydecanoate (5-HD), and N-{[3-(aminomethyl)phenyl]methyl}ethanimidamide (1400W), and 2,7-dichlorodihydro fluorescent diacetate (DCFH-DA) were purchased from Sigma–Aldrich Co (St Louis, MO). HMR-1098 was obtained from Aventis Pharma Deutschland Gmbh (Frankfurt, Germany). Calpain activity assay kit was provided by Promega Corporation (Madison, WI). Inducible nitric oxide synthase (iNOS) antibody, endothelial nitric oxide synthase (eNOS) antibody, and β-actin antibody were purchased from Cell Signaling (Danvers, MA). Bid antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Krebs–Henseleit (KH) solution (pH 7.4) consists of: NaCl 118.0 mmole/L, KCl 4.7 mmole/L, K2PO4 1.2 mmole/L, MgSO4 1.2 mmole/L, NaHCO3 25.0 mmole/L, CaCl2 1.25 mmole/L, and glucose 10.0 mmole/L. Celsior solution (pH 7.4): NaOH 100 mmole/L, KCl 15 mmole/L, MgCl2 13 mmole/L, CaCl2 0.25 mmole/L, mannitol 60 mmole/L, lactobionate 80 mmole/L, histidine 30 mmole/L, and glutamate 20 mmole/L.

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In Vitro Hypothermic Heart Preservation Model

Hearts from male SD rats were rapidly excised and washed in cold (4°C) KH solution to remove blood and then were perfused reversely with KH solution (37°C, gassed with 95% O2 and 5% CO2) on the Langendorff perfusion apparatus. After balancing for 30 minutes, the heart rate (HR), left ventricular end-diastolic pressure (LVEDP), and left ventricular systolic pressure were recorded as the basal values. The left ventricular developed pressure (LVDP) was calculated as left ventricular systolic pressure-LVEDP. Celsior solution (4°C) was perfused into the aorta to induce cardiac arrest. The perfusion time was less than 3 minutes, and the heart surface was cooled simultaneously. After preservation in Celsior solution (4°C) for 9 hours, the hearts were reperfused with KH solution (37°C) for another 60 minutes. Coronary flow (CF) was recorded during the equilibration and reperfusion.

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Lactate Dehydrogenase and Creatine Kinase Contents in the CF

The CF was collected at 5 minutes of reperfusion. The lactate dehydrogenase (LDH) and creatine kinase (CK) levels in the CF were measured by an autonomous biochemical analyzer (CX-4; Beckman Co).

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Measurement of ATP

Intracellular ATP content of the myocardium was measured using a commercially available luciferin–luciferase assay kit (Beyotime, Jiangshu, China). The bioluminescence intensity as a reflection of ATP quantity was determined using a microplate reader (Infinite M200; TECAN, Switzerland).

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Isolation of Cardiac Mitochondria

Cardiac mitochondria were prepared using a protocol as previously described.11 Briefly, rat ventricular myocardium was washed and homogenized in an ice-cold isolation buffer containing 50 mmole/L sucrose, 200 mmole/L mannitol, 5 mmole/L KH2PO4, 1 mmole/L EGTA, 5 mmole/L 3-(N-morpholino) propanesulfonic acid, and 0.1% bovine serum album (pH 7.4). Then samples were centrifuged at 1000g for 10 minutes. The supernatant was further centrifuged at 10,000g for 20 minutes. The pellet was resuspended in isolation buffer (without EGTA) and spun again at 10,000g for another 20 minutes. The final pellet resuspended in the isolation buffer was used for assay.

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Measurement of Reactive Oxygen Species Formation

The level of reactive oxygen species (ROS) in the cardiac mitochondria was determined using DCFH-DA.12 Briefly, 10 μL of mitochondria (10 μg) was suspended in 170 μL HEPES buffer in 96-well plates; 20 μL of 100 μM DCFH-DA was added to each well for a final volume of 200 μL. After incubated at 37°C for 20 minutes, the fluorescence intensity was measured at 485 nm for excitation and 530 nm for emission by a microplate reader (Infinite M200; TECAN). The production of ROS is expressed as fluorescence intensity in relative to control group.

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Assessment of Cell Apoptosis

Cells undergoing apoptotic death present a typical morphology and can be quantified in histopathological tissue sections by TUNEL.7 The cardiac apex of the left ventricle was fixed in 10% formaldehyde and paraffin embedded. After routinely dewaxed and hydrated, the slides of ventricles were incubated with a fluorescein-ligated dUTP and terminal deoxynucleotidyl transferase mixture for 60 minutes at 37°C, and then incubated with horseradish peroxidase solution for 30 minutes at 37°C. Finally, 3,3′-diaminobenzidine color developed, and slides were observed under an optical microscope. Ten visual fields were randomly selected on each slide (total of 500 cells), and cells with brown staining of the nucleus were regarded as apoptotic. The apoptosis index (AI) = apoptotic cell number/total cell number × 100%.

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Calpain Activity Measurement

The calpain activity was measured by using peptide Suc-LLVY-aminoluciferin as a substrate following the instruction of the Promega Technical Bulletin. Briefly, the left ventricle myocardium was homogenized in an ice-cold Tris-buffered saline solution [containing 20 mmole/L Tris-HCl (pH 7.3), 1 mmole/L EGTA, 150 mmole/L NaCl, and 1% Triton ×-100], and centrifuged at 16,000g for 15 minutes. The supernatant was added to a 96-well plate containing Suc-LLVY-Glo reagent at a ratio of 1:1. After gentle mixing for 30 seconds, the contents of the wells were incubated at room temperature for 20 minutes. The luminescence was recorded as relative light units on a plate-reading luminometer. The calpain activity was then normalized to the total protein of sample and expressed as percentage of control.

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Western Blot

Left ventricular myocardium was homogenized in RIPA buffer (containing 50 mmole/L Tris-HCl, 150 mmole/L NaCl, 1 mmmole/L EDTA, 1% Triton ×100, 1% sodium deoxycholate, 0.1% SDS, 1 mmmole/L PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, pH 7.4). After centrifugation at 12,000g for 30 minutes at 4°C, the supernatant was collected. Aliquots of the total cell extract containing 20 μg of protein were loaded on SDS-PAGE. The proteins were blotted onto a nitrocellulose membrane (Invitrogen, Carlsbad, CA). The membrane was incubated with primary antibodies (1:1000) overnight at 4°C. After thorough washing, the membrane was incubated with the horseradish peroxidase–conjugated secondary antibody (1:2000) for 1 hour at room temperature. An enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, AL) was applied to detect the target protein. The membrane was stripped and reprobed with β-actin antibody for the internal loading controls. The band density was determined by Quality One Software (Bio-Rad, Hercules, CA) and normalized to that of β-actin.

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Cervical Heterotopic Heart Transplantation

A cervical heterotopic heart transplantation was performed by using the cuff method.13 The somatic heparinization (300 U/kg) was performed through the inferior vena cava, 2 minutes later the right superior vena cava was ligated and cut off at the distal side of the ligation with a segment of the suture left. The first branch of the aorta was ligated with a 6-0 silk suture, and the suture was retained to the first branch of the aorta. The donor heart was then stored in Celsior solution (4°C). The recipient was anesthetized as described for the donor and laid supine. After a right longitudinal incision parallel to the neck was made, the right common carotid artery and external jugular vein were isolated from the surrounding tissues. The right superior vena cava of donor and external jugular vein of recipient were anastomosed end-to-end by using the cuffing techniques. The anastomosis method between the donor aorta and the common carotid artery of recipient was the same as that used for the vein. The clamp on the jugular vein was removed first after the anastomosis, followed by the clamp on the carotid artery. The donor's heart became red and began to beat right after restoration of blood flow. Serum CK and LDH were measured in recipient rat to analyze the degree of heart injury.

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Animal Grouping

Rat hearts were divided into the following groups (n = 8): (1) control group: did not preserve in Celsior solution, (2) Celsior group: preserved in Celsior solution, (3) Lev groups: preserved in Celsior solution containing levosimendan (10−8, 10−7, or 10−6 mole/L), (4) Lev + 5-HD group: stored in Celsior solution containing levosimendan (10−6 mole/L) and a mitochondrial ATP-sensitive potassium (mitoKATP) channel blocker 5-HD (100 μmole/L), (5) Lev + HMR group: stored in Celsior solution containing levosimendan (10−6 mole/L) and a sarcolemmal ATP-sensitive potassium (sarcKATP) channel blocker HMR-1098 (40 μmole/L), (6) 5-HD/HMR group: stored in Celsior solution containing 5-HD (100 μmole/L) or HMR-1098 (40 μmole/L), (7) Lev + 1400W group: preserved in Celsior solution containing levosimendan (10−6 mole/L) and a selective iNOS inhibitor 1400W (10 μmole/L), and (8) 1400W group: preserved in Celsior solution containing 1400W (10 μmole/L).

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Statistical Analysis

Data were expressed as mean ± SE. The results were analyzed by 1-way analysis of variance and Newman–Keuls Multiple Comparison Test, or a Student's t test using Prism 5.0 (GraphPad Software, San Diego, CA).

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RESULTS

Effect of Levosimendam on Hypothermic Preservation-induced Cardiac Dysfunction and Apoptosis

There was no difference in LVEDP, LVDP, HR, and CF among these groups before hypothermic preservation. The LVEDP increased, and the LVDP, HR, and CF recovery rate of isolated rat heart significantly decreased after 9 hours of hypothermic preservation followed by 60 minutes of reperfusion (P < 0.01). When compared with the Celsior group, supplement Celsior solution with levosimendan (10−7 and 10−6 mole/L) declined the LVEDP and enhanced the LVDP, HR, and CF recovery rate (P < 0.05, Fig. 1).

FIGURE 1

FIGURE 1

Meanwhile, the LDH and CK content in the CF at 5 minutes of reperfusion increased in Celsior group, which was inhibited by administration Celsior solution with levosimendan (10−7 and 10−6 mole/L). Intracellular ATP level was reduced, mitochondrial ROS production and AI increased in cardiomyocytes after 9 hours of hyperthemic preservation. The recovery of ATP content, decrease in ROS, and AI were observed in levosimendan-treated groups (P < 0.05; Fig. 2).

FIGURE 2

FIGURE 2

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Effect of Levosimendam on the Calpain Activity and Bid Protein

Compared with the control group, the calpain activity increased and the cleavage of Bid occurred in rat hearts suffered from 9 hours of hypothermic preservation followed by 60 minutes of reperfusion (P < 0.01). Celsior solution supplement with levosimendan (10−7 or 10−6 mole/L) inhibited the hypothermic preservation-induced activation of calpain and cleavage of Bid (P < 0.01; Fig. 3).

FIGURE 3

FIGURE 3

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Cardiaoprotection Effect of Levosimendam Was Abolished by 5-HD

Neither mitoKATP channel blocker 5-HD nor sarcKATP channel blocker HMR-1098 alone had any effect on hypothermic preservation-induced cardiac injury. However, 5-HD but not HMR-1098 prevented cardiac protection of levosimendan (P < 0.01; Figs. 4, 5). 5-HD also abolished the inhibitory effect of levosimendan on hypothermic preservation-induced activation of calpain, cleavage of Bid (P < 0.01; Fig. 6).

FIGURE 4

FIGURE 4

FIGURE 5

FIGURE 5

FIGURE 6

FIGURE 6

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Cardiaoprotection Effect of Levosimendam Was Abolished by iNOS Inhibitor

Levosimendam (10−6 mole/L) induced increase of myocardial iNOS but not eNOS expression (Fig. 7). A selective iNOS inhibitor 1400W prevented improvement effect of levosimendam on cardiac function and abolished the inhibitory effect of levosimendan on hypothermic preservation-induced activation of calpain, cleavage of Bid, and apoptosis (P < 0.05, Figs. 8–10).

FIGURE 7

FIGURE 7

FIGURE 8

FIGURE 8

FIGURE 9

FIGURE 9

FIGURE 10

FIGURE 10

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Evaluation of Preserved Hearts Transplanted Into the Right Cervical Region

The donor hearts were transplanted heterotopically into the right cervical region. The success rates were 83% and 75% in the heart-grafts preserved in Celsior solution with or without levosimendan, respectively (n = 12). Three hours after transplantation, the LDH and CK release were significantly lower in levosimendan group (LDH: 142.5 ± 21.4 IU/L; CPK: 435.4 ± 52.9 IU/L) than that in Celsior only group (LDH: 255.3 ± 28.6 IU/L; CPK: 795.8 ± 98.58 IU/L).

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DISCUSSION

Myocardial contractile function recovery during reperfusion after hypothermic preservation is depressed. Many studies have been done to change the formula of heart preservation solutions, which show protective effects on preservation hearts.11,14 Levosimendan is a calcium sensitizer approved for use in cardiac, and there have been growing evidence of beneficial effects of levosimendan in I/R injury. Levosimendan can mimic ischemic preconditioning, attenuates reperfusion injury, improve cardiac function, and decrease infarct size in animal models.7,15 Administration of levosimendan during reperfusion protects myocardium against I/R injury in hypothermic global ischemia rat hearts.16 The results of current study further provided evidence that Celsior solution supplement with levosimendan was capable of improving cardiac function recovery in hypothermic preservation rat hearts, indicating a possible application for levosimendan in clinical situations.

Apoptosis is an important cause of early contractile dysfunction after heart transplantation or cardiac ischemia.17,18 The proapoptostic Bcl-2 family protein Bax/Bak activation was initially reported to be a key step essential for mitochondrial dysfunction and apoptosis. But recent studies have shown that Bax activation is not sufficient to induce cell apoptosis. Cleavage of Bid seems prerequisite to the proapoptotic activity of Bax.19 Cleavage of Bid is a proapoptotic factor during long-term hypothermic heart preservation.11 During apoptosis, Bid can be cleaved not only by caspase-8 during death receptor apoptotic signaling but also by other caspases, granzyme B, calpains, and cathepsins.20 Levosimendan has been found to exert antiapoptotic and anti-inflammatory effects in an experimental heart I/R model,21 hypertensive Dahl/Rapp rats,22 and patients with decompensated heart failure.23 Apoptosis mediators, such as soluble Fas and Fas ligand, are reduced significantly after levosimendan administration. The proinflammatory cytokines can modulate cardiac functions by depressing myocardial contractility, and promoting cardiomyocyte apoptosis.24 This study showed that the AI increased after 9 hours of hypothermic preservation followed by 60 minutes of reperfusion. Celsior solution supplement with levosimendan reduced the myocytes apoptosis by inhibiting hypothermic preservation-induced calpain activation and cleavage of Bid.

Levosimendan's cardioprotective effect is mainly mediated by calcium sensitization of contractile proteins, which enhances myocardial performance without changes in oxygen consumption. Studies in muscle strips from the human heart and animal models show that levosimendan induces a moderate increase in intracellular calcium through phosphodiesterase inhibition, but not at therapeutic concentration.25 Levosimendan is not only a calcium sensitizer with positive inotropic but also a vascular KATP channels agonist.26 The mitoKATP channel is an important mediator and end-effector of cardioprotection, and NO, which produced by activation of NOS, is a trigger for the opening of mitoKATP channel.27–29 However, whether the NOS and mitoKATP channel are involved in the cardioprection of levosimendan is still unknown. Recent studies showed that treatment with levosimendan had cardioprotective effects against I/R injury through iNOS activation and KATP channel opening in animal heart models30,31 and against oxidative stress-induced apoptosis in H9c2 cells.32 In this study, it was the mitoKATP channel blocker but not sarcKATP channel blocker that abolished the inhibitory effect of levosimendan on hypothermic preservation-induced cleavage of Bid and apotosis. Levosimendan induced an increase in iNOS protein expression, and the selective iNOS inhibitor prevented cardioprotection effect of levosimendan. The data suggest that levosimendan supplement could reduce myocytes apoptosis in hypothermic preservation heart by activation of iNOS and opening of mitoKATP channels.

Besides that, Levosimendan can also exert protection against liver or kidney I/R injuries in some transplant models.33,34 Grossini et al examined the protective effects of levosimendan against liver oxidative stress in anesthetized rats and found that levosimendan administration counteracted oxidative damages and apoptosis. Levosimendan can bring protection against liver ischemic damages through mechanisms related to NO production and mitoKATP channels function. The potential use of levosimendan in surgery and transplantation is an interesting challenge that remains unmet.

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CONCLUSIONS

In summary, Celsior solution supplement with levosimendan improved cardiac function recovery and reduced myocyte apoptosis in hypothermic preservation rat hearts. Inhibition of cleavage of Bid through iNOS and opening of mitoKATP channel might be involved in the underlying mechanism. The findings of this article are purely speculative, and more data will be needed in future to confirm the clinical advantages of levosimendan in transplant models and in clinical trials.

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Keywords:

hypothermic preservation; inducible nitric oxide synthase; levosimendan; mitochondrial ATP-sensitive potassium channel

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