246 Original Article
Atorvastatin Reduces the Myocardial Content of Coenzyme Q10 in Isoproterenol-induced Heart Failure in Rats
Affiliations
Key words
▶ atorvastatin ● ▶ heart failure ● ▶ coenzyme Q10 ● ▶ malondialdehyde ●
received 26.06.2013 accepted 12.09.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1357178 Published online: October 23, 2013 Drug Res 2014; 64: 246–250 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. Garjani Department of Pharmacology Faculty of Pharmacy Tabriz University of Medical Sciences Daneshgah Street Tabriz – Iran Tel.: + 98/411/3341 315 Fax: + 98/411/3344 798 [email protected]
S. Andalib1, A. Shayanfar2, A. Khorrami1, N. Maleki-Dijazi1, A. Garjani1 1 2
Department of Pharmacology & Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Department of Medical Chemistry, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
▼
The present study was aimed to study the effects of different doses of atorvastatin on Co Q10 content in the myocardium tissue in rats. A subcutaneous injection of isoproterenol (5 mg/kg/day) for 10 days was used for the induction of heart failure. Rats were randomly assigned to control, treatment with atorvastatin (5, 10, 20 mg/ kg/day) and treatment with atorvastatin plus coenzyme Q10 (10 mg/kg/day). Coenzyme Q10 content of myocardium was measured using HPLC method with UV detector after hemodynamic parameters measurements. The malondialdehyde (MDA) content of the myocardium was evaluated in order to determine coenzyme Q10 antioxidative effect. A high dose of atorvas-
Introduction
▼
Despite progresses in treatment and control of heart failure, morbidity and mortality from the disease is still high. Statins have been shown to reduce morbidity and mortality in cardiovascular diseases such as atherosclerosis and coronary artery events [1]. Statins have many effects beyond lipid-lowering that make them of potential benefit in patients with heart failure. They facilitate nitric oxide (NO) synthesis and improve endothelial function, both are typically impaired in patients with heart failure [2]. Experimental studies suggest that statins are beneficial in heart failure as they reduce oxidative stress and inhibit inflammatory responses and myocardial hypertrophy [3]. Statins may have antihypertrophic, antioxidant and antifibrotic effects directly on the myocardium [4] and may suppress immune function and cell proliferation independently from their lipid lowering effect [5]. Although statins prevent important causative factors in many cardiovascular disorders, their beneficial
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
tatin (20 mg/kg/day) was significantly reduced the myocardium content of coenzyme Q10 as compared with isoproterenol treated group (p < 0.001). Compared with atorvastatin alone treated animals, co-administration of coenzyme Q10 with atorvastatin was improved the level of coenzyme Q10 in the myocardium (p < 0.05, p < 0.001). Increasing the dose of atorvastatin also led to increase in MDA content of the myocardium (p < 0.01). Serum lipid profile showed no changes in atorvastatin treated groups. The results of this study demonstrate that high doses of atorvastatin reduce coenzyme Q10 content of the myocardium and increase lipid peroxidation in myocardium which is reversed by coenzyme Q10 co-administration.
effects in heart failure have been questioned [6]. Statins inhibit 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate, this catalysis constituting a rate-limiting step. Mevalonate is the main precursor of coenzyme Q10 (Co Q10) and therefore inhibition of its production by statins blocks Co Q10 synthesis. Decrease in Co Q10 levels caused by statins might have deleterious consequences in patients with severe heart failure [7], because Co Q10 is well defined as a crucial component of the oxidative phosphorylation process converting the energy in carbohydrates and fatty acids into adenosine triphosphate (ATP) in mitochondria [8]. Co Q10 is most abundant in the myocardium and represents an essential component of the mitochondrial respiratory chain that given this tissue’s extreme energy requirements [9]. Another fundamental characteristic of Co Q10 is its antioxidant property [10]. Considering beneficial and antioxidative effects of Co Q10, inhibition of its synthesis by high doses of statins might have detrimental effects on skeletal or
Downloaded by: University of Georgia Libraries. Copyrighted material.
Authors
Original Article 247
Materials and Methods
▼
Animals Male Wistar rats (220 ± 20 g) were used in this study. The animals were given food and water ad libitum. They were housed in the Animal House of Tabriz University of Medical Sciences at a controlled ambient temperature of 25 ± 2 °C with 50 ± 10 % relative humidity and a 12-h light/12-h dark cycle. The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (National Institutes of Health Publication No 85–23, revised 1985).
Chemical and reagents Atorvastatin was a generous gift from Sobhan Pharmaceutical Inc (Tehran-Iran). Isoproterenol was bought from Sigma Chemicals Co (USA), while Co Q10 was purchased from Nature’s bounty Pharmaceutical Inc (USA). The butylhydroxytoluene (BHT), sodium dodecylsulfate (SDS) and thiobarbituric acid (TBA) were obtained from Merck (Germany). Other reagents were of a commercial analytical grade.
Experimental protocol The animals were randomized into 9 groups each consisting of 6 rats. Rats in group 1 (control) received oral administration of normal saline for 30 days and a subcutaneous injection of physiological saline (0.5 mL) for 10 days. Rats in group 2 received oral administration of normal saline for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. Rats in groups 3–5 were pretreated orally, using gastric gavages, with atorvastatin (5, 10 and 20 mg/kg respectively) for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. Rats in group 6 were given Co Q10 (10 mg/kg) orally for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. Rats in groups 7–9 were pretreated orally with atorvastatin (5, 10 and 20 mg/kg; respectively) plus 10 mg/kg Co Q10 for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. All the rats were made to fast overnight. However, they had free access to water until the last administration of the drug.
This helped to measure hemodynamic parameters such as left ventricular end-diastolic pressure (LVEDP), maximum and minimum rates of developed left ventricular pressure (LVdP/dtmax and LVdP/dtmin) [13]. All the parameters were continuously recorded using a Powerlab system (AD Instruments, Australia).
Co Q10 measurements After blood collection from port vein in order to determine serum lipid profile, the hearts were dissected and frozen immediately in liquid nitrogen and subsequently transferred in a − 80 °C freezer. Ethanol was added to samples to remove proteins by partition and denature enzymes. 100 mg of freezeclamped tissue was accurately weighed and subsequently homogenized with 1 mL of water with homogenizer. A volume of 50 μL of a solution of BHT in ethanol (10 mg/mL) was added to each sample to prevent auto-oxidation. Samples after addition of 1 mL of SDS 0.1 M and brief homogenization were then transferred to a 10 mL glass tube fitted with a PTFE-lined screw cap. 2 mL of ethanol was added, and the mixture was vortex-mixed for 30 s. Then 2 mL of hexane was added and the tightly capped test tube was vigorously vortex-mixed for 2 min. It was then centrifuged for 5 min at 2 200 rpm and the hexane organic supernatant layer transferred to a small vial. The extraction with hexane was then repeated a second time. The combined extracts were evaporated with an evaporator (Buchi, rotavapor R-215, Switzerland). Samples after reconstitution with 200 μL of mobile phase were injected to HPLC device under following conditions; column C-18 (4.6 × 250 mm, 5 μm), mobile phase ethanol-methanol (80:20), flow rate 1.2 mL/min, UV detector (Knauer, smartline 2500) wave length 275 nm.
Lipid peroxidation assay Lipid peroxidation was estimated by measuring the content of MDA according to the thiobarbituric acid assay [14]. Lipid peroxidation is generally accepted as an indicator of oxidative stress as a result of free radical overproduction and depletion of antioxidant reserves. 500 mg from each heart tissue samples was homogenized in KCl buffer [tissue to buffer ratio, 1:10 w/v]. Subsequently the homogenates were centrifuged at 4 000 rpm at 4 °C for 10 min. Then 3 mL of phosphoric acid and 1 mL of TBA 0.67 % were added to 250 μL of supernatant of each homogenate to analyze the MDA level in tissue samples. After addition of 3 mL of n-Butanol to each sample, they heated in a water bath for 40 min. The absorbance of MDA at 535 and 520 nm of the upper and colored layer was measured spectrophotometrically. The results values are expressed as percentages of the control.
Serum lipids measurements Serum concentrations of total cholesterol (TC), HDL, and triglycerides (TG) were determined by enzymatic colorimetric methods using commercially available kits (Pars Azmoon Ltd, Iran). The assay was performed according to the manufacturer’s instruction. All samples were measured in duplicate. The concentration of LDL was calculated by the following equation: LDL = TC − (HDL + 0.2TG).
Hemodynamic measurements At the end of the experiment, the animals were anesthetized with sodium pentobarbital (60 mg/kg; i.p). The trachea was cannulated for artificial respiration when the rats no longer responded to external stimuli. In order to evaluate the cardiac left ventricular function, a Mikro Tip catheter transducer (Millar Instruments, INC) was advanced to the lumen of the left ventricle.
Statistics Except for LV dP/dtmax and LV dP/dtmin that were presented as mean ± SEM, all the other results are presented as mean ± SD. One way ANOVA was used to make comparisons between the groups. If the ANOVA analysis indicated significant differences, a Least Significant Difference (LSD) post test was performed to
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
cardiac muscles. This mechanism is thought to be involved in toxic myopathy, causes by statins, and might also be relevant in the progress of cardiac muscle failure [11]. Isoproterenol is a synthetic β-adenoreceptor agonist which its subcutaneous injection induces heart failure and suppresses cardiac functions because of myocardial hypertrophy and fibrosis [12]. In the present study, we aimed at investigating the effects of different doses of atorvastatin on Co Q10 content in the myocardium tissue in rats.
248 Original Article
Fig. 2 Left ventricular maximal and minimal rates of pressure increase (LV dP/dtmax; LV dP/ dtmin) in the control group and in the rats treated with isoproterenol (rats with left heart failure), isoproterenol plus atorvastatin, or isoproterenol plus atorvastatin + Co Q10. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. Values are mean ± SEM (n = 6). a p < 0.001 from respective control value, b p < 0.05, c p < 0.01, d p < 0.001 as compared with isoproterenol treated group and e p < 0.001 compared with atorvastatin (20 mg/kg) treated group).
compare the mean values between the treatment groups and control. Any differences between groups were considered significant at p < 0.05. All analyses were performed using the IBM SPSS statistics 19.0 statistical software.
Results
▼
Effects of atorvastatin and Co Q10 on the hemodynamic responses There was an increase of 38 % in the LVEDP in the isoproterenol received rats, thereby indicating left ventricular dysfunction. Compared to ISO group the high dose of atorvastatin at 20 mg/kg caused a further elevation in the LVEDP. However, the low doses of 5 and 10 mg/kg even had a slightly protective effect by tending to reduce the intra ventricular pressure at the end of diastole ▶ Fig. 1). Administration of atorvastatin with doses of 5, 10 and (● 20 mg/kg with coenzyme Q10 improved the left ventricular function by lowering the left ventricular end diastolic pressure from 11 ± 1.5 mm Hg to 3 ± 1, 3.7 ± 1.2 and 4 ± 1.4 mm Hg, respec▶ Fig. 1; p > 0.05).When comtively in rats with heart failure (● pared with the normal control, the rats with left ventricular dysfunction (isoproterenol group) demonstrated a fall in the values of the left ventricular maximal and minimal rates of pres-
▶ Fig. 2). The high sure (LV dP/dtmax; LV dP/dtmin, p < 0.05; ● dose of atorvastatin (20 mg/kg) worsened the isoproterenolinduced left ventricular dysfunction by a further and very sig▶ Fig. 2; p < 0.01) reduction of contractility (LV dP/ nificant (● dtmax) and relaxation (LV dP/dtmin). Co-administration of Co ▶ Fig. 2; p < 0.001) Q10 reversed these reductions very highly (● when compared with the isoproterenol and isoproterenol plus atorvastatin (20 mg/kg) treated groups.
Effect of atorvastatin on the content of Co Q10 in myocardium Co Q10 content of the myocardium was greatly reduced after ▶ Fig. 3). The reduction of Co isoproterenol injection (p < 0.001; ● Q10 level in the failure hearts was significantly exacerbated with the high dose of atorvastatin (20 mg/kg) and co-administration of Co Q10 (10 mg/kg) along with atorvastatin substantially ▶ Fig. 3). (p < 0.001) prevented this reduction (p < 0.05; ●
Effect of atorvastatin and Co Q10 on heart lipid peroxidation Injection of isoproterenol to induce heart failure was associated with a significant increase in the amount of MDA in myocardium from 0.17 nmol/mg in control group to 0.47 nmol/mg in isoprot▶ Fig. 4). The concentration of MDA in erenol group (p < 0.001; ●
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
Fig. 1 Effect of atorvastatin alone or in combination with Co Q10 (orally) on left ventricular end diastolic pressure in rats treated with isoproterenol. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. a p < 0.05 as compared with isoproterenol treated group, b p < 0.01 compared with atorvastatin (20 mg/kg) treated group).
Original Article 249
Fig. 4 MDA concentration in the heart tissue. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. a p < 0.001 from respective control value, b p < 0.001 as compared with all treated groups, c p < 0.001 as compared with Ator 5 mg/kg treated group, d p < 0.001 as compared with Ator 10 mg/kg treated group, e p < 0.001 as compared with Ator 5 mg/kg treated group, f p < 0.001 as compared with Ator 10 mg/kg treated group, g p < 0.001 as compared with Ator 20 mg/kg treated group).
Table 1 Serum lipid profile of control, isoproterenol treated and treatment groups. There was no significant change in serum lipid profile of treatment groups in comparison with control and isoproterenol treated group. Groups N=6 Control Isoproterenol Atorvastatin (5 mg/kg) + Isoproterenol Atorvastatin (10 mg/kg) + Isoproterenol Atorvastatin (20 mg/kg) + Isoproterenol Co Q10 (10 mg/kg) + Isoproterenol Atorvastatin (5 mg/kg) + Co Q10 + Isoproterenol Atorvastatin( 10 mg/kg) + Co Q10 + Isoproterenol Atorvastatin (20 mg/kg) + Co Q10 + Isoproterenol
Mean serum
Mean serum
Mean seum LDL
Mean seum HDL
Cholestrol (mg/dl)
Triglyceride (mg/dl)
(mg/dl)
(mg/dl)
53.6 ± 8.3 55.8 ± 7.7 55.2 ± 6.9 50.6 ± 5.6 53.4 ± 9.2 54 ± 5.5 60.8 ± 6.2 55.8 ± 8.1
53.8 ± 4.5 45.6 ± 2.2 43.8 ± 4.9 46.6 ± 4 48.6 ± 4.9 39 ± 5.3 40.2 ± 6.6 43.2 ± 7
7.4 ± 4.2 8.6 ± 1.2 10.2 ± 1.9 6.4 ± 2.9 9.2 ± 4 11 ± 5.2 12.4 ± 2 11.4 ± 3.5
34.4 ± 5.3 34.2 ± 1 36.4 ± 4 29.8 ± 4.8 35.8 ± 3.5 36.8 ± 8.8 39.2 ± 5.8 35.6 ± 1
There was no significant change in serum lipid profile of treatment groups in comparison with control and isoproterenol treated group
the heart tissues was considerably decreased by all doses of atorvastatin. However, the low dose of 5 mg/kg of atorvastatin had much more lowering effect than the high dose of 20 mg/kg. Further, the anti oxidative effect of atorvastatin, especially the high dose (20 mg/kg), in the mean of reduction of MDA concentration in the failure myocardium was much more profound by adding Co Q10 into the treatment regime.
Effect of atorvastatin and Co Q10 on serum lipid profile There was no significant changes in the serum lipid profile (Total cholesterol and TG) of all treatment groups in comparison with ▶ Table 1). control or isoproterenol injected groups (●
Discussion
▼
This study indicates that atorvastatin had a dose dependent lowering effect on the Co Q10 content of myocardium in rats with heart failure. This effect might describe that atorvastatin at high doses could worsen the heart failure by inhibition of Co Q10 synthesis and therefore deprive heart tissue from enough ATP to contraction. In the present study, Co Q10 significantly improved the hemodynamic parameters and the left ventricular functions in isoproterenol induced heart failure in rats. Left ventricular end diastolic pressure was also lower in the atorvastatin + Co Q10 group, thereby suggesting improvement in the myocardial stiffness.
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
Fig. 3 Co Q10 concentration in the heart tissue. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. a p < 0.001 from respective control value, b p < 0.05, c p < 0.001 as compared with isoproterenol treated group, d p < 0.05 as compared with Ator10 mg/kg treated group, e p < 0.001 as compared with Ator 20 mg/kg treated group).
250 Original Article
Conclusion
▼
The atorvastatin therapy declined Co Q10 content of myocardium and increased lipid peroxidation in myocardium in a dose dependent manner and Co Q10 co-administration was reversed it. High doses of atorvastatin may cause a further decrease in the already low levels of Co Q10 in patients with chronic heart failure. This may exaggerate the left ventricular depression. Therefore, Co Q10 co-administration could decrease side effect by replacing the endogenous levels as well as producing a synergistic action on oxidative stress. This study suggests that the use of Co Q10 along with statins in patients with heart failure can be useful to prevent side effects of statins.
Acknowledgements
▼
The present study was supported by a grant from the Research Vice Chancellors of Tabriz University of Medical Sciences; Tabriz, Iran.
Conflict of Interest
▼
The authors declare that there is no conflict of interest.
References 1 Tamara B, Horwich M. Statin therapy is associated with improved survival in ischemic and non-ischemic heart failure. J Am Coll Cardiol 2004; 43: 642–648 2 Koh KK, Han SH, Quon MJ. Inflammatory markers and the metabolic syndrome. J Diabetes 2000; 14: 96–107 3 van der Harst P, Voors AA, van Gilst WH et al. Statins in the treatment of chronic heart failure: a systematic review. PLoS Med 2006; 3: 333–337 4 Hayashidani S, Tsutsui H, Shiomi T. Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation 2002; 105: 868–873 5 Glynn RJ. Selective prescribing led to overestimation of the benefits of lipid-lowering drugs. J Clin Epidemiol 2006; 59: 819–828 6 Strandberg TE. Lipid-lowering drugs and heart failure: where do we go after the statin trials? Curr Opin Cardiol 2010; 25: 385–393 7 van der Harst P, Böhm M, Wiek H et al. The case for statin therapy in chronic heart failure. Clinical Research in Cardiology 2008; 97: 139–146 8 Hiroshi M, Atsushi N, Kobayashi J et al. Effects of Co Q10 supplementation on plasma lipoprotein lipid, Co Q10 and liver and muscle enzyme levels in hypercholesterolemic patients treated with atorvastatin: A randomized double-blind study. Atherosclerosis 2007; 195: 182–189 9 James J, Nawarskas S. HMG-CoA Reductase Inhibitors and Coenzyme Q10. Cardiology in Review 2005; 13: 76–79 10 Alleva R, Tomasetti M, Bompadre S et al. Oxidation of LDL and their subfractions: kinetic aspects and Co Q10 content. J Mol Aspects Med 1997; 18: 105–112 11 Schaars CF, Stalenhoef AF. Effects of ubiquinone (coenzyme Q10) on myopathy in statin users. Curr Opin Lipidol 2008; 19: 553–557 12 Ojha S, Goyal S, Kumari S et al. Pyruvate attenuates cardiac dysfunction and oxidative stress in isoproterenol-induced cardiotoxicity. Exp Toxicol Pathol 2012; 64: 393–399 13 Garjani A, Wainwright CL, Zeitlin IJ et al. Effects of endothelin-1 and the ETA-receptor antagonist, BQ123, on ischaemic arrhythmias in anaesthetized rats. J Cardiovas Pharmacol 1995; 25: 634–642 14 Kim SY, Lee E, Kim HP et al. A novel cerebroside from lycii fructus preserves the hepatic glutathione redox system in primary cultures of rat hepatocytes. Biological and Pharmaceutical Bulletin 1999; 22: 873–875 15 Willis RA, Folkers K, Tucker JL et al. Lovastatin decreases coenzyme Q levels in rats. Proc Natl Acad Sci 1990; 87: 8928–8930 16 Satoh K, Yamato A, Nakai T et al. Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischemic dog hearts. Br J Pharmacol 1995; 116: 1894–1898 17 Pepe S, Marasco SF, Haas SJ et al. Coenzyme Q10 in cardiovascular disease. J Mitochondrian 2007; 7: 154–167
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
Willis RA et al., showed that lovastatin decreased myocardial Co Q10 levels in rats and supplementation with Co Q10 restored them [15]. Satoh et al., suggested that a lipid-soluble statin might enter myocardial cells and prevent Co Q10 synthesis through the inhibition of HMG-CoA reductase, while a watersoluble statin would not decrease the myocardial Co Q10 level [16]. Considering that atorvastatin is a lipid soluble statin, may decrease myocardium Co Q10 content by this manner, and it’s independent from lipid lowering action of atorvastatin as our results indicated this. Co Q10 is one of the most important compounds of mitochondrial respiratory chain, and also is a high lipid soluble antioxidant [17]. Reduction in ATP production has been showed in the isolated myocardial mitochondria from failing of animal and human [17]. In the failing heart, decrease in mitochondrial energy metabolism is mostly related to malfunction in the activity of the electron transport chain. Furthermore, Co Q10 may be useful in the treatment of heart failure [17]. Co Q10 role in the heart as a potent anti-oxidant was more highlighted when results indicated that MDA concentration as an oxidative stress indicator in myocardium increased dose dependently by atorvastatin administration and this effect reversed by co-administration of Co Q10.
Atorvastatin Reduces the Myocardial Content of Coenzyme Q10 in Isoproterenol-induced Heart Failure in Rats
Affiliations
Key words
▶ atorvastatin ● ▶ heart failure ● ▶ coenzyme Q10 ● ▶ malondialdehyde ●
received 26.06.2013 accepted 12.09.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1357178 Published online: October 23, 2013 Drug Res 2014; 64: 246–250 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. Garjani Department of Pharmacology Faculty of Pharmacy Tabriz University of Medical Sciences Daneshgah Street Tabriz – Iran Tel.: + 98/411/3341 315 Fax: + 98/411/3344 798 [email protected]
S. Andalib1, A. Shayanfar2, A. Khorrami1, N. Maleki-Dijazi1, A. Garjani1 1 2
Department of Pharmacology & Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Department of Medical Chemistry, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
▼
The present study was aimed to study the effects of different doses of atorvastatin on Co Q10 content in the myocardium tissue in rats. A subcutaneous injection of isoproterenol (5 mg/kg/day) for 10 days was used for the induction of heart failure. Rats were randomly assigned to control, treatment with atorvastatin (5, 10, 20 mg/ kg/day) and treatment with atorvastatin plus coenzyme Q10 (10 mg/kg/day). Coenzyme Q10 content of myocardium was measured using HPLC method with UV detector after hemodynamic parameters measurements. The malondialdehyde (MDA) content of the myocardium was evaluated in order to determine coenzyme Q10 antioxidative effect. A high dose of atorvas-
Introduction
▼
Despite progresses in treatment and control of heart failure, morbidity and mortality from the disease is still high. Statins have been shown to reduce morbidity and mortality in cardiovascular diseases such as atherosclerosis and coronary artery events [1]. Statins have many effects beyond lipid-lowering that make them of potential benefit in patients with heart failure. They facilitate nitric oxide (NO) synthesis and improve endothelial function, both are typically impaired in patients with heart failure [2]. Experimental studies suggest that statins are beneficial in heart failure as they reduce oxidative stress and inhibit inflammatory responses and myocardial hypertrophy [3]. Statins may have antihypertrophic, antioxidant and antifibrotic effects directly on the myocardium [4] and may suppress immune function and cell proliferation independently from their lipid lowering effect [5]. Although statins prevent important causative factors in many cardiovascular disorders, their beneficial
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
tatin (20 mg/kg/day) was significantly reduced the myocardium content of coenzyme Q10 as compared with isoproterenol treated group (p < 0.001). Compared with atorvastatin alone treated animals, co-administration of coenzyme Q10 with atorvastatin was improved the level of coenzyme Q10 in the myocardium (p < 0.05, p < 0.001). Increasing the dose of atorvastatin also led to increase in MDA content of the myocardium (p < 0.01). Serum lipid profile showed no changes in atorvastatin treated groups. The results of this study demonstrate that high doses of atorvastatin reduce coenzyme Q10 content of the myocardium and increase lipid peroxidation in myocardium which is reversed by coenzyme Q10 co-administration.
effects in heart failure have been questioned [6]. Statins inhibit 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate, this catalysis constituting a rate-limiting step. Mevalonate is the main precursor of coenzyme Q10 (Co Q10) and therefore inhibition of its production by statins blocks Co Q10 synthesis. Decrease in Co Q10 levels caused by statins might have deleterious consequences in patients with severe heart failure [7], because Co Q10 is well defined as a crucial component of the oxidative phosphorylation process converting the energy in carbohydrates and fatty acids into adenosine triphosphate (ATP) in mitochondria [8]. Co Q10 is most abundant in the myocardium and represents an essential component of the mitochondrial respiratory chain that given this tissue’s extreme energy requirements [9]. Another fundamental characteristic of Co Q10 is its antioxidant property [10]. Considering beneficial and antioxidative effects of Co Q10, inhibition of its synthesis by high doses of statins might have detrimental effects on skeletal or
Downloaded by: University of Georgia Libraries. Copyrighted material.
Authors
Original Article 247
Materials and Methods
▼
Animals Male Wistar rats (220 ± 20 g) were used in this study. The animals were given food and water ad libitum. They were housed in the Animal House of Tabriz University of Medical Sciences at a controlled ambient temperature of 25 ± 2 °C with 50 ± 10 % relative humidity and a 12-h light/12-h dark cycle. The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (National Institutes of Health Publication No 85–23, revised 1985).
Chemical and reagents Atorvastatin was a generous gift from Sobhan Pharmaceutical Inc (Tehran-Iran). Isoproterenol was bought from Sigma Chemicals Co (USA), while Co Q10 was purchased from Nature’s bounty Pharmaceutical Inc (USA). The butylhydroxytoluene (BHT), sodium dodecylsulfate (SDS) and thiobarbituric acid (TBA) were obtained from Merck (Germany). Other reagents were of a commercial analytical grade.
Experimental protocol The animals were randomized into 9 groups each consisting of 6 rats. Rats in group 1 (control) received oral administration of normal saline for 30 days and a subcutaneous injection of physiological saline (0.5 mL) for 10 days. Rats in group 2 received oral administration of normal saline for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. Rats in groups 3–5 were pretreated orally, using gastric gavages, with atorvastatin (5, 10 and 20 mg/kg respectively) for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. Rats in group 6 were given Co Q10 (10 mg/kg) orally for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. Rats in groups 7–9 were pretreated orally with atorvastatin (5, 10 and 20 mg/kg; respectively) plus 10 mg/kg Co Q10 for 30 days and at the 20th day were subcutaneously injected 5 mg/kg of isoproterenol once at an interval of 24 h for 10 consecutive days. All the rats were made to fast overnight. However, they had free access to water until the last administration of the drug.
This helped to measure hemodynamic parameters such as left ventricular end-diastolic pressure (LVEDP), maximum and minimum rates of developed left ventricular pressure (LVdP/dtmax and LVdP/dtmin) [13]. All the parameters were continuously recorded using a Powerlab system (AD Instruments, Australia).
Co Q10 measurements After blood collection from port vein in order to determine serum lipid profile, the hearts were dissected and frozen immediately in liquid nitrogen and subsequently transferred in a − 80 °C freezer. Ethanol was added to samples to remove proteins by partition and denature enzymes. 100 mg of freezeclamped tissue was accurately weighed and subsequently homogenized with 1 mL of water with homogenizer. A volume of 50 μL of a solution of BHT in ethanol (10 mg/mL) was added to each sample to prevent auto-oxidation. Samples after addition of 1 mL of SDS 0.1 M and brief homogenization were then transferred to a 10 mL glass tube fitted with a PTFE-lined screw cap. 2 mL of ethanol was added, and the mixture was vortex-mixed for 30 s. Then 2 mL of hexane was added and the tightly capped test tube was vigorously vortex-mixed for 2 min. It was then centrifuged for 5 min at 2 200 rpm and the hexane organic supernatant layer transferred to a small vial. The extraction with hexane was then repeated a second time. The combined extracts were evaporated with an evaporator (Buchi, rotavapor R-215, Switzerland). Samples after reconstitution with 200 μL of mobile phase were injected to HPLC device under following conditions; column C-18 (4.6 × 250 mm, 5 μm), mobile phase ethanol-methanol (80:20), flow rate 1.2 mL/min, UV detector (Knauer, smartline 2500) wave length 275 nm.
Lipid peroxidation assay Lipid peroxidation was estimated by measuring the content of MDA according to the thiobarbituric acid assay [14]. Lipid peroxidation is generally accepted as an indicator of oxidative stress as a result of free radical overproduction and depletion of antioxidant reserves. 500 mg from each heart tissue samples was homogenized in KCl buffer [tissue to buffer ratio, 1:10 w/v]. Subsequently the homogenates were centrifuged at 4 000 rpm at 4 °C for 10 min. Then 3 mL of phosphoric acid and 1 mL of TBA 0.67 % were added to 250 μL of supernatant of each homogenate to analyze the MDA level in tissue samples. After addition of 3 mL of n-Butanol to each sample, they heated in a water bath for 40 min. The absorbance of MDA at 535 and 520 nm of the upper and colored layer was measured spectrophotometrically. The results values are expressed as percentages of the control.
Serum lipids measurements Serum concentrations of total cholesterol (TC), HDL, and triglycerides (TG) were determined by enzymatic colorimetric methods using commercially available kits (Pars Azmoon Ltd, Iran). The assay was performed according to the manufacturer’s instruction. All samples were measured in duplicate. The concentration of LDL was calculated by the following equation: LDL = TC − (HDL + 0.2TG).
Hemodynamic measurements At the end of the experiment, the animals were anesthetized with sodium pentobarbital (60 mg/kg; i.p). The trachea was cannulated for artificial respiration when the rats no longer responded to external stimuli. In order to evaluate the cardiac left ventricular function, a Mikro Tip catheter transducer (Millar Instruments, INC) was advanced to the lumen of the left ventricle.
Statistics Except for LV dP/dtmax and LV dP/dtmin that were presented as mean ± SEM, all the other results are presented as mean ± SD. One way ANOVA was used to make comparisons between the groups. If the ANOVA analysis indicated significant differences, a Least Significant Difference (LSD) post test was performed to
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
cardiac muscles. This mechanism is thought to be involved in toxic myopathy, causes by statins, and might also be relevant in the progress of cardiac muscle failure [11]. Isoproterenol is a synthetic β-adenoreceptor agonist which its subcutaneous injection induces heart failure and suppresses cardiac functions because of myocardial hypertrophy and fibrosis [12]. In the present study, we aimed at investigating the effects of different doses of atorvastatin on Co Q10 content in the myocardium tissue in rats.
248 Original Article
Fig. 2 Left ventricular maximal and minimal rates of pressure increase (LV dP/dtmax; LV dP/ dtmin) in the control group and in the rats treated with isoproterenol (rats with left heart failure), isoproterenol plus atorvastatin, or isoproterenol plus atorvastatin + Co Q10. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. Values are mean ± SEM (n = 6). a p < 0.001 from respective control value, b p < 0.05, c p < 0.01, d p < 0.001 as compared with isoproterenol treated group and e p < 0.001 compared with atorvastatin (20 mg/kg) treated group).
compare the mean values between the treatment groups and control. Any differences between groups were considered significant at p < 0.05. All analyses were performed using the IBM SPSS statistics 19.0 statistical software.
Results
▼
Effects of atorvastatin and Co Q10 on the hemodynamic responses There was an increase of 38 % in the LVEDP in the isoproterenol received rats, thereby indicating left ventricular dysfunction. Compared to ISO group the high dose of atorvastatin at 20 mg/kg caused a further elevation in the LVEDP. However, the low doses of 5 and 10 mg/kg even had a slightly protective effect by tending to reduce the intra ventricular pressure at the end of diastole ▶ Fig. 1). Administration of atorvastatin with doses of 5, 10 and (● 20 mg/kg with coenzyme Q10 improved the left ventricular function by lowering the left ventricular end diastolic pressure from 11 ± 1.5 mm Hg to 3 ± 1, 3.7 ± 1.2 and 4 ± 1.4 mm Hg, respec▶ Fig. 1; p > 0.05).When comtively in rats with heart failure (● pared with the normal control, the rats with left ventricular dysfunction (isoproterenol group) demonstrated a fall in the values of the left ventricular maximal and minimal rates of pres-
▶ Fig. 2). The high sure (LV dP/dtmax; LV dP/dtmin, p < 0.05; ● dose of atorvastatin (20 mg/kg) worsened the isoproterenolinduced left ventricular dysfunction by a further and very sig▶ Fig. 2; p < 0.01) reduction of contractility (LV dP/ nificant (● dtmax) and relaxation (LV dP/dtmin). Co-administration of Co ▶ Fig. 2; p < 0.001) Q10 reversed these reductions very highly (● when compared with the isoproterenol and isoproterenol plus atorvastatin (20 mg/kg) treated groups.
Effect of atorvastatin on the content of Co Q10 in myocardium Co Q10 content of the myocardium was greatly reduced after ▶ Fig. 3). The reduction of Co isoproterenol injection (p < 0.001; ● Q10 level in the failure hearts was significantly exacerbated with the high dose of atorvastatin (20 mg/kg) and co-administration of Co Q10 (10 mg/kg) along with atorvastatin substantially ▶ Fig. 3). (p < 0.001) prevented this reduction (p < 0.05; ●
Effect of atorvastatin and Co Q10 on heart lipid peroxidation Injection of isoproterenol to induce heart failure was associated with a significant increase in the amount of MDA in myocardium from 0.17 nmol/mg in control group to 0.47 nmol/mg in isoprot▶ Fig. 4). The concentration of MDA in erenol group (p < 0.001; ●
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
Fig. 1 Effect of atorvastatin alone or in combination with Co Q10 (orally) on left ventricular end diastolic pressure in rats treated with isoproterenol. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. a p < 0.05 as compared with isoproterenol treated group, b p < 0.01 compared with atorvastatin (20 mg/kg) treated group).
Original Article 249
Fig. 4 MDA concentration in the heart tissue. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. a p < 0.001 from respective control value, b p < 0.001 as compared with all treated groups, c p < 0.001 as compared with Ator 5 mg/kg treated group, d p < 0.001 as compared with Ator 10 mg/kg treated group, e p < 0.001 as compared with Ator 5 mg/kg treated group, f p < 0.001 as compared with Ator 10 mg/kg treated group, g p < 0.001 as compared with Ator 20 mg/kg treated group).
Table 1 Serum lipid profile of control, isoproterenol treated and treatment groups. There was no significant change in serum lipid profile of treatment groups in comparison with control and isoproterenol treated group. Groups N=6 Control Isoproterenol Atorvastatin (5 mg/kg) + Isoproterenol Atorvastatin (10 mg/kg) + Isoproterenol Atorvastatin (20 mg/kg) + Isoproterenol Co Q10 (10 mg/kg) + Isoproterenol Atorvastatin (5 mg/kg) + Co Q10 + Isoproterenol Atorvastatin( 10 mg/kg) + Co Q10 + Isoproterenol Atorvastatin (20 mg/kg) + Co Q10 + Isoproterenol
Mean serum
Mean serum
Mean seum LDL
Mean seum HDL
Cholestrol (mg/dl)
Triglyceride (mg/dl)
(mg/dl)
(mg/dl)
53.6 ± 8.3 55.8 ± 7.7 55.2 ± 6.9 50.6 ± 5.6 53.4 ± 9.2 54 ± 5.5 60.8 ± 6.2 55.8 ± 8.1
53.8 ± 4.5 45.6 ± 2.2 43.8 ± 4.9 46.6 ± 4 48.6 ± 4.9 39 ± 5.3 40.2 ± 6.6 43.2 ± 7
7.4 ± 4.2 8.6 ± 1.2 10.2 ± 1.9 6.4 ± 2.9 9.2 ± 4 11 ± 5.2 12.4 ± 2 11.4 ± 3.5
34.4 ± 5.3 34.2 ± 1 36.4 ± 4 29.8 ± 4.8 35.8 ± 3.5 36.8 ± 8.8 39.2 ± 5.8 35.6 ± 1
There was no significant change in serum lipid profile of treatment groups in comparison with control and isoproterenol treated group
the heart tissues was considerably decreased by all doses of atorvastatin. However, the low dose of 5 mg/kg of atorvastatin had much more lowering effect than the high dose of 20 mg/kg. Further, the anti oxidative effect of atorvastatin, especially the high dose (20 mg/kg), in the mean of reduction of MDA concentration in the failure myocardium was much more profound by adding Co Q10 into the treatment regime.
Effect of atorvastatin and Co Q10 on serum lipid profile There was no significant changes in the serum lipid profile (Total cholesterol and TG) of all treatment groups in comparison with ▶ Table 1). control or isoproterenol injected groups (●
Discussion
▼
This study indicates that atorvastatin had a dose dependent lowering effect on the Co Q10 content of myocardium in rats with heart failure. This effect might describe that atorvastatin at high doses could worsen the heart failure by inhibition of Co Q10 synthesis and therefore deprive heart tissue from enough ATP to contraction. In the present study, Co Q10 significantly improved the hemodynamic parameters and the left ventricular functions in isoproterenol induced heart failure in rats. Left ventricular end diastolic pressure was also lower in the atorvastatin + Co Q10 group, thereby suggesting improvement in the myocardial stiffness.
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
Fig. 3 Co Q10 concentration in the heart tissue. (Iso: isoproterenol; Ator: atorvastatin; Q10: Co Q10 10 mg/kg. a p < 0.001 from respective control value, b p < 0.05, c p < 0.001 as compared with isoproterenol treated group, d p < 0.05 as compared with Ator10 mg/kg treated group, e p < 0.001 as compared with Ator 20 mg/kg treated group).
250 Original Article
Conclusion
▼
The atorvastatin therapy declined Co Q10 content of myocardium and increased lipid peroxidation in myocardium in a dose dependent manner and Co Q10 co-administration was reversed it. High doses of atorvastatin may cause a further decrease in the already low levels of Co Q10 in patients with chronic heart failure. This may exaggerate the left ventricular depression. Therefore, Co Q10 co-administration could decrease side effect by replacing the endogenous levels as well as producing a synergistic action on oxidative stress. This study suggests that the use of Co Q10 along with statins in patients with heart failure can be useful to prevent side effects of statins.
Acknowledgements
▼
The present study was supported by a grant from the Research Vice Chancellors of Tabriz University of Medical Sciences; Tabriz, Iran.
Conflict of Interest
▼
The authors declare that there is no conflict of interest.
References 1 Tamara B, Horwich M. Statin therapy is associated with improved survival in ischemic and non-ischemic heart failure. J Am Coll Cardiol 2004; 43: 642–648 2 Koh KK, Han SH, Quon MJ. Inflammatory markers and the metabolic syndrome. J Diabetes 2000; 14: 96–107 3 van der Harst P, Voors AA, van Gilst WH et al. Statins in the treatment of chronic heart failure: a systematic review. PLoS Med 2006; 3: 333–337 4 Hayashidani S, Tsutsui H, Shiomi T. Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation 2002; 105: 868–873 5 Glynn RJ. Selective prescribing led to overestimation of the benefits of lipid-lowering drugs. J Clin Epidemiol 2006; 59: 819–828 6 Strandberg TE. Lipid-lowering drugs and heart failure: where do we go after the statin trials? Curr Opin Cardiol 2010; 25: 385–393 7 van der Harst P, Böhm M, Wiek H et al. The case for statin therapy in chronic heart failure. Clinical Research in Cardiology 2008; 97: 139–146 8 Hiroshi M, Atsushi N, Kobayashi J et al. Effects of Co Q10 supplementation on plasma lipoprotein lipid, Co Q10 and liver and muscle enzyme levels in hypercholesterolemic patients treated with atorvastatin: A randomized double-blind study. Atherosclerosis 2007; 195: 182–189 9 James J, Nawarskas S. HMG-CoA Reductase Inhibitors and Coenzyme Q10. Cardiology in Review 2005; 13: 76–79 10 Alleva R, Tomasetti M, Bompadre S et al. Oxidation of LDL and their subfractions: kinetic aspects and Co Q10 content. J Mol Aspects Med 1997; 18: 105–112 11 Schaars CF, Stalenhoef AF. Effects of ubiquinone (coenzyme Q10) on myopathy in statin users. Curr Opin Lipidol 2008; 19: 553–557 12 Ojha S, Goyal S, Kumari S et al. Pyruvate attenuates cardiac dysfunction and oxidative stress in isoproterenol-induced cardiotoxicity. Exp Toxicol Pathol 2012; 64: 393–399 13 Garjani A, Wainwright CL, Zeitlin IJ et al. Effects of endothelin-1 and the ETA-receptor antagonist, BQ123, on ischaemic arrhythmias in anaesthetized rats. J Cardiovas Pharmacol 1995; 25: 634–642 14 Kim SY, Lee E, Kim HP et al. A novel cerebroside from lycii fructus preserves the hepatic glutathione redox system in primary cultures of rat hepatocytes. Biological and Pharmaceutical Bulletin 1999; 22: 873–875 15 Willis RA, Folkers K, Tucker JL et al. Lovastatin decreases coenzyme Q levels in rats. Proc Natl Acad Sci 1990; 87: 8928–8930 16 Satoh K, Yamato A, Nakai T et al. Effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on mitochondrial respiration in ischemic dog hearts. Br J Pharmacol 1995; 116: 1894–1898 17 Pepe S, Marasco SF, Haas SJ et al. Coenzyme Q10 in cardiovascular disease. J Mitochondrian 2007; 7: 154–167
Andalib S et al. Atorvastatin Reduces Coenzyme Q10 of Heart … Drug Res 2014; 64: 246–250
Downloaded by: University of Georgia Libraries. Copyrighted material.
Willis RA et al., showed that lovastatin decreased myocardial Co Q10 levels in rats and supplementation with Co Q10 restored them [15]. Satoh et al., suggested that a lipid-soluble statin might enter myocardial cells and prevent Co Q10 synthesis through the inhibition of HMG-CoA reductase, while a watersoluble statin would not decrease the myocardial Co Q10 level [16]. Considering that atorvastatin is a lipid soluble statin, may decrease myocardium Co Q10 content by this manner, and it’s independent from lipid lowering action of atorvastatin as our results indicated this. Co Q10 is one of the most important compounds of mitochondrial respiratory chain, and also is a high lipid soluble antioxidant [17]. Reduction in ATP production has been showed in the isolated myocardial mitochondria from failing of animal and human [17]. In the failing heart, decrease in mitochondrial energy metabolism is mostly related to malfunction in the activity of the electron transport chain. Furthermore, Co Q10 may be useful in the treatment of heart failure [17]. Co Q10 role in the heart as a potent anti-oxidant was more highlighted when results indicated that MDA concentration as an oxidative stress indicator in myocardium increased dose dependently by atorvastatin administration and this effect reversed by co-administration of Co Q10.
