Effects of Levosimendan on Hydrogen Peroxide Induced Contraction in Human Saphenous Vein

doi:10.4236/pp.2011.24030

Paper Menu >>

Journal Menu >>

Pharmacology & Pharmacy, 2011, 2, 233-237

doi:10.4236/pp.2011.24030 Published Online October 2011 (http://www.SciRP.org/journal/pp)

233

Effects of Levosimendan on Hydrogen Peroxide

Induced Contraction in Human Saphenous Vein

Burak Cem Soner1, Ayse Saide Sahin1*, Ipek Duman2, Niyazi Gormus3

1Department of Pharmacology, Meram Faculty of Medicine, Selcuk University, Konya, Turkey; 2Pharmacology, Ministry of Health,

Konya, Turkey; 3Department of Cardiovascular Surgery, Meram Faculty of Medicine, Selcuk University, Konya, Turkey.

Email: *[email protected]

Received May 30th, 2011; revised July 28th, 2011; accepted August 5th, 2011.

ABSTRACT

Aim: Increased oxidative stress plays important roles in vascular dysfunction in patients undergoing coronary artery

bypass graft surgery. Hydrogen peroxide (H2O2) is used as an experimental model for oxidative stress. The present

study was designed to assess the effects of levosimendan pretreatment on the contractile effects induced by H2O2 in hu-

man saphenous vein (HSV) segments. Methods: We studied H2O2 induced contractions of isolated HSV mounted in

standard tissue baths. H2O2 (10–6 - 10–3 M) was added cumulatively to the organ bath. Concentration-response curves

to H2O2 were repeated in the presence of levosimendan (10–8 M). In the second series of experiments, strips were con-

tracted with 5-HT (10–5 M). When the contraction reached a stable plateau, H2O2 was administrated cumula tively into

the organ bath. The same procedure was conducted in the presence of levosimendan. Results: Pretreatment of the SV

strips with levosimendan significantly reduced the contractile response to each concentration of H2O2. 5-HT produced

contractions in SV strips. Further treatment of these strips with H2O2 resulted in statistically significant concentra-

tion-dependent increases in tension. Preincubation of the tissues with levosimendan did not significantly influence the

maximum amplitude of the 5-HT-induced tone but inh ibited the contra ctile effect of H2O2 on th e 5-HT-induced contrac-

tion. Conc lus ion : Pretreatment of HSV with clinical concentrations of levosimendan inhibits the vasoconstriction

caused by oxidative stress, indicating its potential preventive effect against oxidative stress induced graft spasm.

Keywords: Hy dro ge n Peroxide, Human Saphenous Vein, Levosimendan

1. Introduction

Human saphenous vein is frequently used for coronary

artery bypass grafting because of ready availability and

suppleness [1]. Vasospasm of the graft following coro-

nary artery bypass graft (CABG) surgery is a major prob-

lem and may cause perioperative and late failure of by-

pass conduits. There are ongoing researches on deter-

mining the mechanisms causing vasospasm and various

vasodilators have been studied to prevent or reverse

vasoconstriction in various grafts [2,3].

Increased oxidative stress plays important roles in

myocardial and vascular dysfunction in patients under-

going CABG [4]. Hydrogen peroxide (H2O2) is an im-

portant derivative of oxidative metabolism and is a major

contributor in oxidative stress-induced functional and

metabolic dysfunction [5]. Although generation of H2O2

may occur under normal physiological conditions and is

not restricted to pathological conditions, its formation in

the endothelium of blood vessels under stress conditions

such as diabetes, hypertension, preeclampsia and extra-

corporeal membrane oxygenation holds an important

place in CABG surgery [6]. H2O2 is already used as an

experimental model for oxidative stress and it has been

shown to induce concentration dependent increases in

contraction in various blood vessels including saphenous

veins [7,8] and potentiates the effects of vasoconstrictor

agents [9].

Levosimendan is a new cardiac enhancer that exerts

positive inotropic effects on heart failure by calcium sen-

sitization of contractile proteins. Recent research has

displayed that levosimendan also causes peripheral vaso-

dilatation and may also have antioxidant properties [10].

The present study was designed to assess the effects of

levosimendan pretreatment on the contractile effects of

H2O2 in human saphenous vein (HSV) segments.

2. Methods

The study was approved by the ethics committee of Sel-

cuk University, Meram Faculty of Medicine and in-

Effects of Levosimendan on Hydrogen Peroxide Induced Contraction in Human Saphenous Vein

234

formed consent was obtained from all patients undergo-

ing myocardial revascularization surgery. The discarded

HSV segments were placed in cold Krebs-Henseleit so-

lution (KHS: NaCl 119 mmol/L, KCl 4.7 mmol/L,

MgSO4 1.5 mmol/L, KH2PO4 1.2 mmol/L, CaCl2 2.5

mmol/L, NaHCO3 25 mmol/L, and glucose 11 mmol/L)

and transported to the laboratory for the study in 20 min-

utes. After removal of the surrounding tissue, vein seg-

ments were cut into helical 12 - 15 mm strips and sus-

pended in a 20 ml organ bath containing KHS at 37˚C

and continuously bubbled with 95% O2 and 5% CO2 gas

mixture. Tissues were gradually stretched to a basal rest-

ing tension of 1.0 g and were allowed to equilibrate to

their own resting tension for 60 minutes, and during this

period the KHS was changed every 15 minutes.

After an equilibration period, strips (n = 8) were con-

tracted using 10–5 M serotonin (5-HT). To standardize

responses between strips, once maximal 5-HT contrac-

tions were recorded and used as a standard by which

subsequent contractions of the tissue could be expressed

(as a percentage of this contraction).

Two types of experiment were performed. In the first

set of experiments, the effect of H2O2 was investigated in

levosimendan-pretreated strips (n = 8). First the maxi-

mum contractions to 5-HT (10 - 5 M) were recorded in

HSV strips. Tissues were then washed twice within 15

minutes of washout intervals and thereafter concentra-

tion-response curves to cumulative H2O2 (10–6 - 10–3 M)

were recorded. After two 15 minutes of washout intervals

concentration-response curves to cumulative H2O2 (10–6 -

10–3 M) were repeated in the presence of levosimendan

(10–8 M).

In the second set of experiments, the effects of levosi-

mendan on oxidative stress caused by H2O2 in the tissues

contracted with 5-HT were investigated. Strips (n = 8)

were contracted with 5-HT (10–5 M). When the contrac-

tion reached a stable plateau, H2O2 was administrated

cumulatively (10–6 - 10–3 M) into the organ baths in one-

log increments. The same procedure was also conducted

in the presence of levosimendan (10–8 M).

In all groups, isometric recording of tension changes

were obtained with force transducers (Grass FTO4; Grass

Instrument Co, W. Warwick, RI, USA) connected through

amplifiers to a polygraph (Grass 7D).

The effects of H2O2 are expressed as the percentage of

the control contractile response elicited at 10–5 M 5-HT.

The maximum contraction (Emax) and the concentration

required to achieve 50% of maximum contraction (EC50)

were calculated for H2O2.

All data were expressed as mean ± s.e. mean (SEM).

The statistical significance of differences among groups

was analyzed by using unpaired student’s t-test. For all

studies, p value less than 0.05 was considered significant.

All the drugs were prepared freshly at the day of the

study. The following compounds were used: Hydrogen

peroxide (H2O2) obtained from Merck, Darmstadt, Ger-

many; levosimendan, Simdax, from Abbott Laboratories;

serotonin (5-HT) from Sigma, St. Louis, MO, USA.

3. Results

The effects of H2O2 (10–6 - 10–3 M) on HSV strips in the

absence and presence of levosimendan are summarized

in Figure 1. A maximum contraction of 32.4% ± 1.12%

was obtained with H2O2 10–3 M and the EC50 value was

4.6 × 10–6 ± 0.3 M. Pretreatment of the HSV strips with

levosimendan (10–8 M) significantly reduced the contrac-

tile response to each one concentration of H2O2 (p <

0.05). The EC50 value for H2O2 was 4.5 × 10–5 ± 0.2 and

Emax was 16.0% ± 1.0% in the presence of levosimendan.

5-HT (10–6 M) produced contractions in HSV strips.

Further treatment of strips with H2O2 (10–6 - 10–3 M) re-

sulted in statistically significant concentration-dependent

increases in tension (Emax: 21.0 ± 2.2; EC50: 2.5 × 10–6 ±

0.17). Preincubation of the tissues with levosimendan did

not significantly influence the maximum amplitude of the

5-HT-induced tone but inhibited the contractile effect of

H2O2 on the 5-HT-induced contraction (Emax: 2.67 ± 0.33)

(Figure 2).

4. Discussion

Results from our present in vitro model for oxidative

stress shows that:

1) Pretreatment with clinical concentration of levosi-

mendan (10–8 M) attenuates the contractile response elic-

Figure 1. Concentration-response curves for contraction of

HSV elicited by H2O2 under control conditions and after

incubation with 10–8 M levosimendan. Each point repre-

sents the mean ± SEM expressed as percentage of the ten-

sion induced by 10–5 M 5-HT (n = 8).

Effects of Levosimendan on Hydrogen Peroxide Induced Contraction in Human Saphenous Vein235

Figure 2. Concentration-response curves for H2O2 on the

vasoconstriction induced by 10 –5 M 5-HT in HSV under con-

trol conditions and after incubation with 10–8 M levosimen-

dan. Each point represents the mean ± SEM expressed as

percentage of the tension induced by 10–5 M 5-HT (n = 8).

ited by H2O2.

2) Pretreatment with levosimendan (10–8 M) prevents

contractile responses caused by H2O2 in the tissues pre-

contracted with 5-HT but does not affect 5-HT elicited

contractions.

H2O2 is found in the human plasma at micromolar

concentrations and its concentration can increase up to

millimolar levels in pathological states including myo-

cardial ischemia and heart failure [11]. It can diffuse

from its site of formation, easily crossing cell membranes

and producing cellular oxidative damage [6]. However,

previous studies have suggested that H2O2 may be an

important mediator in the vasculature as a regulator of

vasomotor tone [12]. In previous studies, 50 mM to 10

mM concentrations of H2O2 were used to induce experi-

mental oxidative stress [13-15]. In the present study, we

used 10–6 - 10–3 M concentrations of H2O2. In order to

evaluate the effect of levosimendan on vasoconstriction

during oxidative stress, first we tested the effect of

levosimendan with incubation on cumulative H2O2 con-

centration response curve in HSV strips at resting tension.

Our results showed that the application of levosimendan

inhibited contractions of H2O2 in HSV strips indicating

its potential preventive effect against the graft spasm

induced by oxidative stress. Active contraction induced

by 5-HT is considered to be an important pathological

mechanism for inducing arterial spasm and may cause

perioperative and late failure of bypass conduits [1].

Since H2O2 also potentiates contractile responses to

various agents [9], after 5-HT induced contractions we

evaluated the effect with the same concentration of levo-

simendan incubation on cumulative H2O2 concentration

response curve. Treatment of these strips with H2O2 po-

tentiated contraction induced by 5-HT. Levosimendan

10–8 M prevented contractile responses caused by H2O2

in the tissues precontracted with 5-HT but does not affect

5-HT elicited contractions.

Plasma level of levosimendan during clinical practice

is important to mention for proper interpretation of the

experimental results. A single 0.5 mg oral dose of levo-

simendan produces a peak plasma concentration of 20

ng/ml (0.07 micromol) in patients with congestive heart

failure [16]. Vasodilatation by levosimendan is believed

to be achieved at a higher plasma concentration when

compared to its positive inotropic effect. In patients with

ischemic heart disease, 0.25 mg and 0.5 mg levosimen-

dan increased left ventricular function, but a significant

decrease in total peripheral resistance was seen only after

2 mg and 4 mg doses [17]. In the present study, the se-

lected concentration (10–8 M) of levosimendan is elected

as used for its inotropic effect. This concentration, which

significantly inhibited the contractions to H2O2 in HSV

strips, is much lower than previous in vitro studies,

which reported that higher concentrations of levosimen-

dan (in the micromolar range) required to elicit vasodila-

tion. Mirkhani et al. [16] reported a maximum relaxation

of 45.4% with 10–4 M of levosimendan in norepineph-

rine-induced contraction. Similar to our result with 5-HT

induced contraction, they observed no relaxation with

10–8 M of levosimendan in HSV precontracted with nore-

pinephrine. In another study, levosimendan at a concen-

tration of 10–8 M did not effect saphenous vein precon-

tracted with 5-HT and the maximum relaxation induced

by levosimendan (3 × 10–6 M) was 28.1 ± 7.5% with an

EC50 of 0.32 ± 0.04 microM [17].

The present study design does not include the mecha-

nisms of by which levosimendan attenuates H2O2 induced

contractions or cause vasodilation. Previous studies have

shown that levosimendan causes positive inotropic and

antistunning effects on the heart. This effect was shown to

be mediated by calcium sensitization of contractile pro-

teins and vasodilator and antiischemic effects mediated by

the opening of ATP-sensitive potassium channels in vas-

cular smooth muscle cells [18]. Levosimendan reduces

plasma levels of malondialdehyde, a marker of oxidative

stress, in patients with heart failure [19], inhibits H2O2

induced cardiomyocytes apoptotic cell death by activating

KATP channels [20] and preconditioning with levosimen-

dan prevents contractile dysfunction due to H2O2-induced

oxidative stress in human myocardium [21].

Although levosimendan has been shown to induce

vasodilatation in human radial and internal mammary

arteries and in HSV [22-24], this is the first study con-

sidering the effect of levosimendan on H2O2-induced con-

Effects of Levosimendan on Hydrogen Peroxide Induced Contraction in Human Saphenous Vein

236

traction in HSV.

In conclusion, according to our results we may specu-

late that pretreatment of HSV with clinical concentra-

tions of levosimendan inhibit the vasoconstriction caused

by oxidative stress, indicating its potential preventive

effect against oxidative stress induced graft spasm. Fur-

ther research is warranted to assess the exact mechanisms

responsible for this effect of levosimendan.

REFERENCES

[1] E. Nakamura, N. Tanaka N, M. Kuwabara M, A. Yama-

shita, Y. Matsuo, T. Kanai, T. Onitsuka, Y. Asada, H.

Hisa and R. Yamamoto, “Relative Contributions of 5-Hy-

droxytryptamine (5-HT) Receptor Subtypes in 5-HT-In-

duced Vasoconstriction of the Distended Human Saphe-

nous Vein as a Coronary Artery Bypass Graft,” Biologi-

cal & Pharmaceutical Bulletin, Vol. 34, No. 1, 2011, pp.

82-86. doi:10.1248/bpb.34.82

[2] C. A. Hamilton, G. O’Dowd, L. McIntosh, G. Berg, J. But-

ler, V. Pathi, R. Williams, J. L. Reid and A. F. Dominiczak,

“Vasorelaxant Properties of Isolated Human Radial Arter-

ies: Comparison with Internal Mammary Arteries,” Athe-

rosclerosi s, Vol. 160, No. 2, 2002, pp. 345-353.

doi:10.1016/S0021-9150(01)00605-0

[3] S. Attaran, L. John and A. El-Gamel, “Clinical and Po-

tential Use of Pharmacological Agents to Reduce Radial

Artery Spasm in Coronary Artery Surgery,” The Annals

of Thoracic Surgery, Vol. 85, No. 4, 2008, pp. 1483-1489.

doi:10.1016/j.athoracsur.2007.10.042

[4] J. T. Parissis, I. Andreadou, S. L. Markantonis, V. Bistola,

A. Louka, A. Pyriochou, I. Paraskevaidis, G. Filippatos, E.

K. Iliodromitis and D. T. Kremastinos, “Effects of Levo-

simendan on Circulating Markers of Oxidative and Ni-

trosative Stress in Patients with Advanced Heart Failure,”

Atherosclerosis, Vol. 195, No. 2, 2007, pp. 210-215.

doi:10.1016/j.atherosclerosis.2007.07.011

[5] K. H. Konz, M. Haap, K. E. Hill, R. F. Burk and R. A.

Walsh, “Diastolic Dysfunction of Perfused Rat Hearts

Induced by Hydrogen Peroxide. Protective Effect of Se-

lenium,” Journal of Molecular and Cellular Cardiology,

Vol. 21, No. 8, 1989, pp. 789-795.

doi:10.1016/0022-2828(89)90718-9

[6] M. A. Rodriguez-Martinez, E. C. Garcia-Cohen, A. B.

Baena, R. Gonzalez, M. Salaices and J. Marin, “Contrac-

tile Responses Elicited by Hydrogen Peroxide in Aorta

from Normotensive and Hypertensive Rats. Endothelial

Modulation and Mechanism Involved,” British Journal of

Pharmacology, Vol. 125, No. 6, 1998, pp.1329-1335.

doi:10.1038/sj.bjp.0702200

[7] Z. W. Yang, T. Zheng, A. Zhang, B. T. Altura and B. M.

Altura, “Mechanisms of Hydrogen Peroxide-Induced Con-

traction of Rat Aorta,” European Journal of Pharmacol-

ogy, Vol. 344, No. 2-3, 1998, pp. 169-181.

doi:10.1016/S0014-2999(97)01576-8

[8] A. S. Sahin, E. A. Atalik, T. K. Sahin and N. Doğan,

“Cooling and Response to Hydrogen Peroxide in Human

Saphenous Vein: Role of the Endothelium,” Fundamental

& Clinical Pharmacology, Vol. 19, No. 3, 2005, pp. 341-

346. doi:10.1111/j.1472-8206.2005.00330.x

[9] K. Watanabe, Y. Okatani and Y. Sagara, “Potentiating

Effect of Hydrogen Peroxide on the Serotonin-Induced

Vasocontraction in Human Umbilical Artery,” Acta Ob-

stetricia et Gynecologica Scandinavica, Vol. 75, No. 9,

1996, pp. 783-789. doi:10.3109/00016349609054704

[10] J. T. Parissis, I. Andreadou, V. Bistola, I. Paraskevaidis,

G. Filippatos and D. T. Kremastinos, “Novel Biologic

Mechanisms of Levosimendan and Its Effect on the Fail-

ing Heart,” Expert Opinion on Investigational Drugs, Vol.

17, No. 8, 2008, pp. 1143-1150.

doi:10.1517/13543784.17.8.1143

[11] R. S. Barlow, A. M. El-Mowafy and R. E. White, “H2O2

Opens BKca Channels via the PLA2-Arachidonic Acid

Signaling Cascade in Coronary Artery Smooth Muscle,”

American Journal of Physiology: Heart and Circulatory

Physiology, Vol. 279, No. 2, 2000, pp. H475-H483.

[12] T. Suvorava, N. Lauer, S. Kumpf, R. Jacob, W. Meyer

and G. Kojda, “Endogenous Vascular Hydrogen Peroxide

Regulates Arteriolar Tension in Vivo,” Circulation, Vol.

112, No. 16, 2005, pp. 2487-2495.

doi:10.1161/CIRCULATIONAHA.105.543157

[13] A. Hara, T. Suızuki, H. Hashizume, N. Shishido, M. Na-

kamura, F. Ushikubi and Y. Abiko, “Effects of CP-060S,

a Novel Ca2+ Channel Blocker, on Oxidative Stress in

Cultured Cardiac Myocytes,” European Journal of Phar-

macology, Vol. 385, No. 1, 1999, pp. 81-88.

doi:10.1016/S0014-2999(99)00708-6

[14] S. Shimizu, Y. Saitoh, T. Yamamoto and K. Momose,

“Stimulation by Hydrogen Peroxide of L-Arginine Me-

tabolism to L-Citrulline Coupled with Nitric Oxide Syn-

thesis in Cultured Endothelial Cells,” Research Commu-

nications in Chemical Pathology Pharmacology, Vol. 84,

No. 3, 1994, pp. 315-329.

[15] Z. W. Yang, T. Zheng, J. Wang, A. Zhang, B. T. Altura

and B. M. Altura, “Hydrogen Peroxide Induces Contraction

and Raises [Ca2+]i in Canine Cerebral Arterial Smooth

Muscle: Participation of Cellular Signaling Pathways,”

Naunyn-Schmiedeberg’s Archives of Pharmacology, Vol.

360, No. 6, 1999, pp. 646-653.

doi:10.1007/s002109900128

[16] H. Mirkhani, M. Shafa and H. Khazraei, “Comparison of

the Effects of Levosimendan and Papaverine on Human

Internal Mammary Artery and Saphenous Vein,” Car-

diovascular Drugs and Therapy, Vol. 23, No. 5, 2009, pp.

355-359. doi:10.1007/s10557-009-6197-0

[17] E. P. Sandell, M. Hayha, S. Antila, P. Heikkinen, P. Ot-

toila, L. A. Lehtonen and P. J. Pentikainen, “Pharmaco-

kinetics of Levosimendan in Healthy Volunteers and Pa-

tients with Congestive Heart Failure,” Journal of Car-

diovascular Pharmacology, Vol. 26, Supplement 1, 1995,

pp. 557-562.

[18] G. Lilleberg, S. Sundberg and M. S. Nieminen, “Dose-

range Study of a New Calcium Sensitizier, Levosimendan,

in Patients with Left Ventricular Dysfunction,” Journal of

Cardiovascular Pharmacology, Vol. 26, Supplement 1,

1995, pp. S563-S569.

Effects of Levosimendan on Hydrogen Peroxide Induced Contraction in Human Saphenous Vein

237

[19] O. Pollesello and Z. Papp, “The Cardioprotective Effects

of Levosimendan: Preclinical and Clinical Evidence,”

Journal of Cardiovascular Pharmacology, Vol. 50, No. 3,

2007, pp. 257-263. doi:10.1097/FJC.0b013e3180986230

[20] C. Avgeropoulou, I. Andreadou, S. Markantonis-Kyrou-

dis, M. Demopoulou, P Missovoulos, A. Androulakis and

I. Kallikazaros, “The Ca2+-Sensitizer Levosimendan Im-

proves Oxidative Damage, BNP and Pro-Inflammatory

Cytokine Levels in Patients with Advanced Decompen-

sated Heart Failure in Comparison to Dobutamine,” Euro-

pean Journal of Heart Failure, Vol. 7, No. 5, 2005, pp.

882-887. doi:10.1016/j.ejheart.2005.02.002

[21] D. M. Kopustinskiene, P. Pollesello and N. E. Saris, “Levo-

simendan is a Mitochondrial KATP Channel Opener,” Euro-

pean Journal of Pharmacology, Vol. 428, No. 3, 2001, pp.

311-314. doi:10.1016/S0014-2999(01)01350-4

[22] A. S. Sahin, N. Gormus and A. Duman, “Preconditioning

with Levosimendan Prevents Contractile Dysfunction

Due to H2O2-Induced Oxidative Stress in Human Myo-

cardium,” Journal of Cardiovascular Pharmacology, Vol.

50, No. 4, 2007, pp. 419-423.

doi:10.1097/FJC.0b013e318123fbf9

[23] J. Pataricza, J. Szolnoky, I. Krassoi, Z. Hegedus, A. Kun,

A. Varro and J. G. Papp, “Vazorelaxing Effect of Levosi-

Mendan against 5-Hydroxytryptamine-Induced Contrac-

tions in Isolated Human Conduit Bypass Grafts,” Journal

of Pharmacy and Pharmacology, Vol. 58, No. 8, 2006, pp.

1107-1112. doi:10.1211/jpp.58.8.0012

[24] J. Hohn, J. Pataricza and A. Petri, “Levosimendan Interacts

with Potassium Channel Blockers in Human Saphenous

Veins,” Basic & Clinical Pharmacology & Toxicology, Vol.

94, No. 6, 2004, pp. 271-273.