Key words

Jacek Owczarek, Magdalena Jasińska, Urszula Kurczewska,

Daria Orszulak-Michalak

The Influence of Concomitant Administration

of Simvastatin at Different Doses with Metoprolol

on Heart Rate and Blood Pressure in

Normo-cholesterolemic and HyperNormo-cholesterolemic Rats*

Wpływ łącznego podania simwastatyny w różnych dawkach

z metoprololem na częstość rytmu serca oraz ciśnienie tętnicze

u szczurów z zaburzeniami i bez zaburzeń lipidowych

Department of Biopharmacy, Medical University of Lodz, Poland

Abstract

Background. β1-adrenergic blocking drugs and inhibitors of HMGCo-A reductase (statins) are widely used by

a lot of patients in the prevention of cardiovascular events. Previous studies have shown that the administration of statins influences β adrenergic signaling.

Objectives. The aim of the study was to evaluate the influence of a chronic 4-week administration of simvastatin given at different doses with concomitant metoprolol on heart rate and blood pressure.

Material andMethods. The experiments were performed in normotensive Wistar rats. The animals were divided into 2 groups: normocholesterolemic and hypercholesterolemic. During the 4 weeks, hypercholesterolemic rats received a diet with 5% cholesterol and 2,5% cholic acid. Drugs: metoprolol 30 mg × kg–1 _{b.w.; simvastatin 1 mg ×}

× kg–1 _{b.w.; simvastatin 20 mg × kg}–1 _{b.w.; simvastatin 1 mg × kg}–1 _{b.w. + metoprolol 30 mg × kg}–1 _{b.w., or}

simvas-tatin 20 mg × kg–1 _{b.w. + metoprolol 30 mg kg}–1 _{b.w. were given intragastrically over the 4-week period. For the}

evaluation of heart rate and blood pressure, the signals were provided by an Isotec pressure transducer.

Results. Concomitant administration of simvastatin at 1 and 20 mg × kg–1 _{b.w with metoprolol in}

normocholester-olemic rats statistically decreased heart rate as compared to the control group and the group receiving simvastatin alone. No statistical differences between the groups receiving simvastatin at dose 1 mg/kg–1 _{with metoprolol and}

simvastatin at dose 20 mg/kg–1 _{with metoprolol (381.64 ± 7.90 vs. 388.21 ± 5,15 1/min p = 0.05) were observed. In}

the hypercholesterolemic animals, the heart rate evaluation after concomitant administration of simvastatin given at different doses with metoprolol showed comparable statistical implications as compared to normocholester-olemic rats. Concomitant administration of simvastatin in different doses on normo- and hypercholesternormocholester-olemic rats did not significantly influence blood pressure.

Conclusions. Nosignificant impact ofchronic administration of simvastatin given at different doses to normo- and hypercholesterolemic Wistar rats receiving metoprolol on the heart rate was observed (Adv Clin Exp Med 2011, 20, 1, 39–45).

Key words: rats, simvastatin, metoprolol, heart rate.

Streszczenie

Wprowadzenie. Leki blokujące receptory β1-adrenergiczne oraz inhibitory reduktazy 3-hydroksy

3-metyloglutary-lo-koenzymu A (statyny) są powszechnie stosowane u pacjentów w zapobieganiu incydentom sercowo-naczynio-wym. Wcześniejsze badania wykazały, że podawanie statyn wpływa na sygnalizację β-adrenergiczną.

Cel pracy. Ocena wpływu podawania simwastatyny w różnych dawkach na wartości rytmu serca i ciśnienia tętni-czego po łącznym 4-tygodniowym podawaniu z metoprololem.

Materiał i metody. Badanie zostało przeprowadzone na szczurach Wistar o prawidłowym ciśnieniu krwi. Zwierzęta

Adv Clin Exp Med 2011, 20, 1, 39–45 ISSN 1230-025X

OrIGINAL PAPErS

© Copyright by Wroclaw Medical University

Cholesterol-loweringdrugs such as inhibitors of 3-hydroxy 3-methylglutaryl-coenzyme A re-ductase (HMG-CoA, statins), are widely used by a lot of patients in the prevention of cardiovascular events [1, 2]. It is connected with the LDL-choles-terol lowering activities as well as the pleiotropic effects of statins [3, 4]. Lowering criteria including both LDL-cholesterol and total cholesterol blood level in patients with cardiovascular disease re-quires higher doses of statins for optimization of hypolipemic therapy. β1-adrenergic blocking drugs

are recommended for reduction of myocardial ischemia and prevention of angina pectoris. They have been shown to decrease mortality and sudden death particularly by reduction resting heart rate and in this case by reduction of myocardial oxygen consumption in patients with ischemic heart dis-ease and heart failure [5–8]. Previous studies have shown that statins might influence β-adrenergic signaling [9] although in our studies, two-week administration of simvastatin in different doses to normocholesterolemic rats did not modify meto-prolol injection-induced depressed heart rate and does not modify metoprolol’s impact on blood pressure [10, 11]. The aim of the study was to evaluate the influence of simvastatin given at dif-ferent doses on heart rate and blood pressure with concomitant chronic 4-week administration of metoprolol.

Material and Methods

Animals

The study was approved by the Ethics Com-mittee of the Medical University of Lodz (Po-land) – 43/ŁB405/2008. The experiments were performed on 101, anesthetized Wistar rats, out-bred males. A several-day adaptation period was

scheduled prior to the beginning of the experi-ment. After the adaptation period, the animals were divided into 2 groups: normocholesterolemic and hypercholesterolemic. Normocholesterolemic rats received a normal diet during the 4 weeks. Hypercholesterolemic rats received a normal diet with 5% cholesterol and 2,5% cholic acid during the 4 weeks. After this period, each group was di-vided into 6 subgroups, rats receiving, respective-ly: group I: placebo (0.2% methylcellulose), group II: metoprolol 30 mg × kg–1 _{b.w.; group III:}

sim-vastatin 1 mg × kg–1 _{b.w.; group IV: simvastatin}

20 mg × kg–1 _{b.w.; group V: simvastatin 1 mg ×}

kg–1 _{b.w. + metoprolol 30 mg × kg}–1 _{b.w.; group VI:}

simvastatin 20 mg × kg–1 _{b.w. + metoprolol 30 mg}

× kg–1 _{b.w. The drugs were given intragastrically}

(i.g.) over the four-week period. Animals had free access to diet (normal or hypercholesterolemic) and water. After the drug administration period, heart rate and hemodynamic parameters were measured. Surgery was performed 24 hours after administration of the last drug dose and 10 hours after the last food supply. For the subsequent sur-gical procedures, anesthesia was initiated by an in-traperitoneal (i.p.) dose of pentobarbital sodium at 60 mg × kg–1 _{b.w. The anesthesiawas maintained}

by intraperitoneal bolus injections of pentobarbi-tal sodium at 10 mg × kg–1 _{b.w. as needed. For the}

measurement of the heart rate (Hr) and arterial blood pressure (mean (MAP), systolic (SAP) and diastolic (DAP)), a catheter was implanted into the right carotid artery. The signals were provided by an Isotec pressure transducer connected to a direct current bridge amplifier (Hugo Sachs Elektronik). Analog signals were amplified and recorded on a computer via an A/D converter (HSE Haemo-dyn software for Microsoft Windows 95/98/NT) and were evaluated according to the algorithms. The values of MAP, SAP and DAP were calculated automatically by the software during the measure-były podzielone na 2 grupy: z prawidłowym stężeniem cholesterolu i podwyższonym stężeniem cholesterolu. Szczury z grupy z podwyższonym stężeniem cholesterolu otrzymywały dietę wzbogaconą 5% zawartością chole-sterolu i 2,5% zawartością kwasu cholowego. Leki: metoprolol 30 mg × kg–1 _{m.c.; simwastatyna 1 mg × kg}–1 _m.c.;

simwastatyna 20 mg × kg–1 _{m.c.; simwastatyna 1 mg × kg}–1 _{m.c. + metoprolol 30 mg × kg}–1 _{m.c., simwastatyna}

20 mg × kg–1 _{m.c. + metoprolol 30 mg × kg}–1 _{m.c. były podawane dożołądkowo przez okres 4 tygodni.}

Wyniki. Łączne podanie simwastatyny w dawkach: 1 i 20 mg × kg–1 _{m.c. z metoprololem szczurom z grupy z}

prawi-dłowym stężeniem cholesterolu znacząco statystycznie zwalniało rytm serca w porównaniu z grupą kontrolną oraz grupy otrzymującej samą simwastatynę. Nie odnotowano istotnej statystycznie różnicy w zakresie rytmu serca mię-dzy osobnikami z grup z prawidłowym stężeniem cholesterolu otrzymującymi różne dawki simwastatyny (1 i 20 mg × kg–1 _{m.c.) łącznie z metoprololem (381,64 ± 7.90 vs 388,21 ± 5,15 1/min p = 0,05). W grupie zwierząt z}

hipercho-lesterolemią ocena wartości rytmu serca po łącznym podaniu różnych dawek simwastatyny (1 i 20 mg × kg–1 _m.c.)

łącznie z metoprololem wykazywała podobną zależność statystyczną. Łączne podanie różnych dawek simwastatyny łącznie z metoprololem nie wpłynęło istotnie statystycznie na wartości ciśnienia tętniczego.

Wnioski. Nie zaobserwowano wpływu przewlekłego podawania simwastatyny w różnych dawkach na zwolnienie rytmu serca u szczurów z prawidłowym i podwyższonym stężeniem cholesterolu otrzymujących metoprolol (Adv Clin Exp Med 2011, 20, 1, 39–45).

ment procedure. For further statistical analysis, the output values of the parameters were calculated as the mean from 3-min periods. After heart rate and blood pressure assessment, 0.25 ml of blood samples were taken for a further lipid profile ex-amination. Surgical procedures and recording of heart rate and blood pressure were provided as described previously [10, 11].

Statistics

All data were presented as means ± SD (stan-dard deviation). Statistical comparisons between baseline conditions and metoprolol injection were done by pairedStudent’s t-test. Comparisons be-tween the groups were performed using ANOVA. Post-hoc comparisons were performed using an LSD test. Normal distribution of parameters was checked by means of a Shapiro-Wilks test. If data were not normally distributed or the values of

vari-ance were different, ANOVA with Kruscal-Wallis and Mann-Whitney’s U tests were used. All pa-rameters were considered statistically significantly different if p < 0.05. The statistical analysis of the heart rate and hemodynamic parameters was per-formed using Statgraphics 5.0 plus software.

Results

Four-week administration of simvastatin alone or concomitant administration with metoprolol to normocholesterolemic rats significantly decreased LDL cholesterol and increased HDL cholesterol level as compared to the control group. A decrease of LDL cholesterol level after administration of simvastatin alone or concomitant administration with metoprolol was observed in hypercholester-olemic rats. The changes in animal lipid profiles are shown in Table 1.

Table 1. Total cholesterol (TCH), HDL cholesterol, LDL cholesterol(LDL), triglycerides (TG) (mean ± SD) in rats fed normo- and hypercholesterolemic diet [mmol/l]

Tabela 1. Cholesterol całkowity (TCH), HDL cholesterol, LDL cholesterol (LDL), triglicerydy (TG) (średnie ± SD) u szczurów na diecie normalnej i hipercholesterolowej [mmol/l]

TCH HDL LDL TG

K_N (N = 10) 1.48 ± 0.12 0.38 ± 0.09 0.95 +/-0.29 0.32 ± 0.13 M_N (N = 9) 1.14 ± 0.08 0.36 ± 0.08 0.89 +/ - 0.17 0.28 ± 0.08 S1_N (N = 10) 1.34 ± 0.21 0.57 ± 0.05* 0.56 ± 0.14* 0.46 ± 0.14 S20_N (N = 7) 1.39 ± 0.14 0.69 ± 0.14* 0.44 ± 0.09* 0.56 ± 0.18 S1_M_N (N = 6) 1.51 ± 0.14 0.60 ± 0.13* 0.62 ± 0.09* 0.58 ± 0.26 S20_M_N (N = 8) 1.32 ± 0.12 0.54 ± 0.18* 0.56 ± 0.10* 0.43 ± 0.15 K_H (N = 10) 8.09 ± 1.53 0.36 ± 0.09 6.25 ± 0.65 3.24 ± 0.62 M_H (N = 9) 7.65 ± 1.15 0.39 ± 0.13 5.38 ± 0.82 3.57 ± 0.15 S1_H (N = 8) 6.35 ± 1.81 0.61 ± 0.12* 4.45 ± 0.21* 2.82 ± 0.57 S20_H (N = 9) 2.01 ± 0.16* 0.42 ± 0.14 1.29 ± 0.92* 0.65 ± 0.64* S1_M_H (N = 8) 7.23 ± 0.66 0.41 ± 0.05 5.08 ± 0.33* 3.47 ± 0.79 S20_M_H (N = 7) 1.32 ± 0.18* 0.33 ± 0.04 0.54 ± 0.03* 0.89 ± 0.23* K_N – normocholesterolemic control group, K_H – hypercholesterolemic control group, M_H – hypercholesterolemic group receiving metoprolol, M_N – normocholesterolemic group receiving metoprolol, S1_N – normocholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S1_H – hypercholesterolemic group receiving simvastatin at dose 1 mg/ kg b.w., S20_N – normocholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S20_H – hypercholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S1_M_N – normocholesterolemic group receiving metoprolol and sim-vastatin at dose 1 mg/kg b.w., S20_M_N – normocholesterolemic group receiving metoprolol and simsim-vastatin at dose 20 mg/kg b.w., S1_M_H – hypercholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_H – hypercholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w. (*) P < 0.05 as compared to the control group.

The heart rate (Hr) in the control groups re-ceiving a normo- and hypercholesterolemic diet was 433.82 ± 22.6 and 418.03 ± 29.05 beats/min (p = 0.05) respectively. The four-week administra-tion of metoprolol to normo and hypercholesterol-emic rats significantly decreased Hr as compared to the control groups (390.76 ± 20.5 and 384.28 ± 21,8 1/min). In groups receiving simvastatin at 1 and 20 mg × kg–1 _{b.w. during the 4 weeks, the Hr}

was comparable to the control rats. No differences between groups receiving simvastatin at 1 and 20 mg/kg–1 _{were observed, as well.}

After concomitant administration of simvas-tatin at 1 and 20 mg × kg–1 _{b.w. with metoprolol}

to normocholesterolemic rats, the Hr statistically decreased as compared to the control rats and the group receiving simvastatin alone. No statistical differences between groups receiving simvastatin at 1 mg/kg–1 _{with metoprolol and simvastatin at 20}

mg/kg–1 _{with metoprolol (381.64 ± 7.90 vs. 388.21 ±}

± 5.15 beats/min; p = 0.05) were denoted.

The Hr after concomitant administration of simvastatin at 1 and 20 mg × kg–1 _{b.w. with}

me-toprolol to hypercholesterolemic rats showed the same statistical implications as compared to nor-mocholesterolemic rats. No statistical differences between hypercholesterolemic groups receiving simvastatin at dose 1 mg/kg–1 _{with metoprolol and}

simvastatin at dose 20 mg/kg–1 _{with metoprolol}

were shown, as well (380.76 ± 21.64 vs 382.20 ± ± 15.98 beats/min) (Fig. 1).

Concomitant administration of simvastatin at 1 and 20 mg × kg–1 _{b.w. with metoprolol in normo-}

and hypercholesterolemic rats did not significantly influence arterial blood pressure. The results of MAP, SAP and DAP are presented in Tab. 2.

Discussion

In our study, concomitant administration of simvastatin given at different doses with meto-prolol did not influence the heart rate as compared to rats receiving metoprolol alone. Similar effects were observed in our previous studies [10, 11]. Conversely, some reports on the influence of

Fig. 1. Mean heart rate [beats/min] in Wistar rats fed normo- and hypercholesterolemic diet

K_N – normocholesterolemic control group, K_H – hyperc-holesterolemic control group, M_H – hyperchyperc-holesterolemic group receiving metoprolol, M_N – normocholesterolemic group receiving metoprolol, S1_N – normocholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S1_H – hypercholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S20_N – normocholesterolemic group receiv-ing simvastatin at dose 20 mg/kg b.w., S20_H – hyperc-holesterolemic group receiving simvastatin at dose 20 mg/ kg b.w., S1_M_N – normocholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_N - normocholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w., S1_M_H – hypercho-lesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_H – hypercholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w. (*) P < 0.05 as compared to the control group. (a) p < 0.05 as compared to the group receiving simvastatin alone

HMG-CoA reductase inhibitors on β-adrenergic receptor activity have been described. It has been shown that statins, by reducing the production of important isoprenoids like farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), could increase β-adrenergic receptor density [9]. The isoprenylation process could influence the membrane association and function of heterotri-meric G-proteins by interfering with γ- subunit (Gγ) isoprenylation [12, 13], especially the iso-prenylation of signaling molecules, including the monomeric GTPases of the rho and ras families. Moreover, the isoprenylation of Gγ has been found to be essential for membrane attachment of Gγ as well as Gβ [14]. Previous studies demonstrated that treatment of cardiac myocytes with a statin reduced cAMP and induced a significant increase in β-adrenergic receptor density and, in this way, statins might have an impact on heart rate. The dose dependent influence of statins on

beta1-adrenergic signaling was studied as well [9]. The clinical manifestations of the interaction of statins with β1-adrenergic blockers on heart rate have not

been elucidated yet. Their concomitant usage does not seem to impact heart rate negatively, nor blood pressure. Concomitant administration of simvas-tatin at 1 and 20 mg × kg–1 _{b.w. with metoprolol}

in normo- and hypercholesterolemic rats did not significantly influence blood pressure. Even so, clinical observations in the UCSD Statin Study and the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA) suggest that HMG-CoA reductase inhibitors might have some blood-pressure-lowering properties in addition to their effect on lipids [15, 16]. On the other hand, the CArE study showed no significant reduction of BP with statin therapy [2]. Different results be-tween the CArE and ASCOT studies may result from the degree of LDL-C reduction achieved in the above trials.The laboratory studies showed the

Table 2. Summary statistics (mean ± SD) for arterial blood pressure [mm Hg] in normo- and hypercholesterolemic rats

Tabela 2. Statystyka opisowa (średnia ± SD) dla wartości ciśnienia tętniczego [mm Hg] u szczurów normo- i hipercholeste-rolowych

N Systolic arterial pressure (SAP) [mm Hg]

Mean

arterial pressure (MAP) [mm Hg]

Diastolic

arterial pressure (DAP) [mm Hg]

K_H 10 106.75 ± 2.39 95.28 ± 3.33 86.32 ± 3.85

K_N 10 106.17 ± 3.19 95.93 ± 3.69 83.65 ± 6.18

M_H 9 107.46 ± 3.53 95.82 ± 4.57 86.86 ± 5.80

M_N 9 106.84 ± 1.30 92.68 ± 5.70 83.89 ± 4.22

S1_H 8 104.39 ± 2.45 94.37 ± 4.85 85.97 ± 5.87

S1_M_H 8 106.81 ± 2.52 93.63 ± 6.38 85.66 ± 4.98 S1_M_N 6 105.23 ± 1.26 91.52 ± 4.10 84.05 ± 4.19 S1_N 10 106.71 ± 3.29 96.18 ± 6.29 86.33 ± 4.18 S20_H 9 105.04 +- 3.05 95.35 ± 8.56 85.46 ± 3.62 S20_M_H 7 105.42 ± 2.89 94.61 ± 6.70 85.69 ± 4.36 S20_M_N 8 104.60 ± 3.93 95.87 ± 4.57 86.67 ± 5.79 S20_N 7 107.26 ± 3.65 95.01 ± 6.80 88.29 ± 6.64

K_N – normocholesterolemic control group, K_H – hypercholesterolemic control group, M_H – hypercholesterolemic group receiving metoprolol, M_N – normocholesterolemic group receiving metoprolol, S1_N – normocholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S1_H – hypercholesterolemic group receiving simvastatin at dose 1 mg/ /kg b.w., S20_N – normocholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S20_H – hypercholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S1_M_N – normocholesterolemic group receiving metoprolol and sim-vastatin at dose 1 mg/kg b.w., S20_M_N – normocholesterolemic group receiving metoprolol and simsim-vastatin at dose 20 /mg/kg b.w., S1_M_H – hypercholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_H – hypercholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w.

link between statins therapy and vascular function and this could explain the beneficial pharmacolog-ical effect of statins in patients with hypertension. Statins improve endothelial function and decrease oxidative stress and inflammation [17–19]. They were shown to reduce the production of reactive oxygen species, suchas superoxide anion, and hy-droxyl radicals, as shown in experimental studies. This action may also contribute to the vasodilatory effects of statins. It has already been established that statins increase the endothelial productionof NO and this effect is correlated with the upregu-lation of endothelialNO synthase expression [20]. It has suggested that the effect described above might be intensified by the simultaneous inhibi-tion of G proteins with reduced endothelialNO synthase mrNA degradation and, thus, increased NO bioavailability [21, 22]. Vascular statin activity

may result from drug impact on the decrease of vasoconstrictor endothelin-1 level [21]. Another possible mechanism of statin’s BP-lowering effect involves the downregulation of the angiotensin II-type 1 receptor.The angiotensin II–type 1 receptor is overexpressed inhypercholesterolemic patients and it may be improvedby the administration of statins,which were also shown to markedly reduce the vasoconstrictor response to angiotensin II in-fusion [24]. reduction of large artery stiffness and blood pressure in normolipidemic patients with isolated systolic hypertension statins could im-prove systemic arterial compliance [25].

The authors concluded that administration of simvastatin in different doses not decrease slowing heart rate after chronic concomitant administra-tion of metoprolol in normo- and hypercholester-olemic rats.

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Address for correspondence:

Jacek Owczarek

Department of Biopharmacy Medical University of Lodz Muszyńskiego 1

90-153 Łódź Poland

E-mail: [email protected]

Conflict of interest: None declared