Pharmacology & Pharmacy, 2011, 2, 233-237
doi:10.4236/pp.2011.24030 Published Online October 2011 (http://www.SciRP.org/journal/pp)
Copyright © 2011 SciRes. 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: *aysesaide@gmail.com
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).
Copyright © 2011 SciRes. PP
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-
Copyright © 2011 SciRes. PP
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.
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