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Statin (Mevalotin) preconditioning decreases infarct size in senile rat myocardial infarction model
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ScienceDirect Journal of Acute Medicine 4 (2014) 127e132 www.e-jacme.com
Original Research
Statin (Mevalotin) preconditioning decreases infarct size in senile rat myocardial infarction model Tzong-Luen Wang a,b,* a
Department of Emergency Medicine, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b Medical School, Fu-Jen Catholic University, Taipei, Taiwan Received 15 July 2014; accepted 28 July 2014 Available online 18 September 2014
Abstract Aim: Although statins have been proved to have cardioprotective effects, it remains to be investigated whether “statin-induced preconditioning (PC)” can reduce the infarct size for senile animals. Methods: Senile Wistar rats (weighing 350e450 g) were randomly assigned to one of 14 experiment groups (n 5 for each group). All hearts underwent 30 minutes of coronary artery occlusion and 2 hours of reperfusion (control procedure). Ischemic PC was elicited by three cycles of 5-minute occlusion and 5-minute reperfusion. Statin PC was performed by administering Mevalotin (1 mg/kg, the optimal dose being determined by a doseeeffect study) 30 minutes prior to the control procedure. In addition, pretreatment with glibenclamide, 5-hydroxydecanoic acid, chelerythrine, genistein, and NG-nitro-4-arginine methyl ester, or their combination, was also performed to test possible signaling mechanisms. Results: Statin PC decreased the infarct size significantly compared with the control group (22 ± 4% vs. 53 ± 4%, p < 0.01). Pretreatment with NG-nitro-4-arginine methyl ester completely blocked the effects of Mevalotin (infarct size: 48 ± 4% vs. 22 ± 4%, p < 0.01). The cardioprotective effects of statin PC could be blunted by antagonism of mitochondrial adenosine triphosphate (ATP)-sensitive potassium and sarcolemmal ATP-sensitive potassium channels (glibenclamide: 50 ± 4% vs. 22 ± 4%, p < 0.01; 5-hydroxydecanoic acid: 50 ± 4% vs. 22 ± 4%, p < 0.01). In addition, protein kinase C antagonist (chelerythrine) and tyrosine kinase antagonist (genisteine) also have similar effects (chelerythrine: 48 ± 4% vs. 22 ± 4%, p < 0.01; genistein: 45 ± 4% vs. 22 ± 4%, p < 0.01). Conclusion: It is established in this study the myocardial PC caused by statins, that is involved cellular mechanisms including mainly the pathway of endothelial nitrous oxide synthase (eNOS)enitrous oxide donationeATP-sensitive potassium channels. Protein kinase C and tyrosine kinase have regulatory effects. Copyright © 2014, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: endothelial nitrous oxide synthase; mitochondrial adenosine triphosphate-sensitive potassium; preconditioning; statins
1. Introduction Preconditioning (PC) involves a pretreatment or premaneuver that is able to decrease myocardial damage after a severe decrease in coronary flow. We have demonstrated many modes of PC in previous experiments to show cardioprotective effects.1e3 The term “preconditioning” means a powerful * Department of Emergency Medicine, Shin-Kong Wu Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei, Taiwan. E-mail address: [email protected].
experimental tool that can reduce the infarct size (IS) after ischemiaereperfusion or other pretreatments such as adrenergic stimulation,4 activation of adenosine receptors5 and adenosine triphosphate (ATP)-sensitive potassium (KATP) channels,6 and induction of heat shock proteins7 and oxidative stress.8 Some investigators have further studied the possible effector cells of such PC. Besides neutrophils, which we earlier proposed to be affected by PC,1 endothelial cells are the main target cells at present.2,9e11 Activation of G protein-linked phospholipase C, tyrosine kinase pathways, and protein kinase C, and generation of
http://dx.doi.org/10.1016/j.jacme.2014.07.001 2211-5587/Copyright © 2014, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved.
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oxygen radicals are usually involved in PC.12 In addition, KATP channels are considered the most possible triggers and effectors of PC.12 Recent studies suggested that mitochondrial KATP channels (MitoKATP channels), in addition to sarcolemmal KATP channels, might account mainly for the protective mechanism.12e16 MitoKATP channel opening has been proved to be related to mitochondrial swelling and optimization of respiration,13 prevention of mitochondrial calcium overload,14 and control of reactive oxygen radicals,15,16 all of which are possibly linked to cardioprotection during severe myocardial ischemia and infarction. As mentioned above, cellular targets of PC seem to be heterogeneous. Although saline-perfused isolated hearts or isolated cardiomyocytes can be preconditioned,17,18 the socalled microvascular component of PC has been presented.19e21 The concept is concerned with the reduction of postischemic inflammation or mitigation of leukocyte adhesion in the perfused vascular bed after PC. Endothelial cells, the interface between parenchymal tissue and blood cells, are involved in the significant pathophysiological process. On the other hand, statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been available for decades. Some clinical trials have indicated that statins appear to have beneficial effects independent of their cholesterollowering properties, when compared with other agents. These so-called pleiotropic properties include antiinflammatory effects, plaque stabilization,22e24 improved endothelial function, and inhibition of vascular smooth muscle cell proliferation.25 The beneficial pleiotropic effects of statins in protecting the isolated murine hearts against lethal reperfusion injury via activation of phosphatidylinositide 3-kinases (PI3K)/Akt prosurvival signaling pathway and its downstream effectors, endothelial nitrous oxide synthase (eNOS), were also demonstrated.26 In addition, phosphorylation of multiple prosurvival pathways involving p44/p42 Mitogen-activated protein kinases (MAPK), p38 MAPK, and heat shock protein (HSP) 27 were also demonstrated in a later study.27 It is well known that statins are associated with nitrous oxide synthase (NOS) activation. Nitrous oxide (NO) is a unique intra/intercellular messenger involved in the regulation of many physiological functions.28 Any compounds that modulate NO release may display a great therapeutic value.29 Different statins such as lovastatin, atorvastatin, pravastatin, and simvastatin demonstrate variable potency to enhance the NO/oxygen concentration ratio by enhancing NOS, resulting in increased endothelial NO activities.30 In addition, acutely administered statins minimize the damaging effect of transient cerebral31 or myocardial ischemia32 in animal models. Many studies31e33 clearly indicate the role of NOS activated by statins. In addition, some other statins have been proved to attenuate oxidant-induced mitochondrial dysfunction in cardiac myocytes.34 Clinical observations also demonstrated significant protection from myonecrosis among patients undergoing atherectomy with statins.35 We designed the following in vivo study to investigate the presence of the phenomenon “statin-induced PC” and its cellular mechanisms.
2. Methods 2.1. Animal preparation Male Wistar rats, weighing 350e450 g and fed a standard diet, were included in this study. After induction of anesthesia with intraperitoneal pentobarbitone (50 mg/kg body weight), all the animals were ventilated with room air via endotracheal intubation and a rodent respirator (Harvard Apparatus, Boston, MA, USA) at 60e70 breaths per minute. The right carotid artery was cannulated to measure the mean blood pressure and heart rate via a Could PE-50 pressure transducer, which was connected to a Grass polygraph (Grass Technologies, RI. USA). The right jugular vein was cannulated to infuse saline or drugs. A left thoracotomy was performed to expose the heart at the fifth intercostal space. After removing the pericardium, a 6-0-Prolene ligation, together with a snare occluder, was placed over the origin of the left anterior descending artery. Regional ischemia was achieved by pulling the snare and securing the threads with a mosquito hemostat, which indicated that approximately 60% of the myocardium was ischemic in our study. 2.2. Experimental protocols The rats included in the present study were randomly assigned to one of the experimental groups (n 5 for each group). All hearts underwent 30 minutes of coronary artery occlusion and 2 hours of reperfusion (control procedure) after an initial 30-minute stabilization period. Ischemic PC was elicited by three cycles of 5-minute occlusion and 5-minute reperfusion. Statin PC was performed by administering the drug Mevalotin (1 mg/kg, the optimal dose being determined by a doseeeffect study) 30 minutes prior to the control procedure. Glibenclamide (2 mg/kg), a sarcolemmal KATP channel antagonist, was administered 30 minutes prior to statin PC to examine the role of the channel. The effect of 5-hydroxydecanoic acid (5-HD), a MitoKATP channel-selective antagonist, was also examined by giving 10 mg/kg 5-HD 5 minutes prior to the statin PC. To evaluate the possible roles of protein kinase C and tyrosine kinase, their antagonists, chelerythrine (2 mg/kg, 30 minutes prior to statin PC) and genistein (5 mg/kg, 5 minutes prior to statin PC), or their combination for synergic effects, were also given as a pretreatment in various groups. To test the role of NOS in statin-induced cardioprotection, we also determined the effects of the NOS inhibitor NG-nitro-4-arginine methyl ester (L-NAME). L-NAME (50 mg/kg) was administered 30 minutes prior to ischemic PC with or without statin pretreatment, and also prior to statin PC. In summary, the rats were assigned to the following groups for carrying out the experiments: 1. Control group: 30-minute occlusion þ 2-hour reperfusion (control procedure) 2. Ischemic PC: (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 3. Statin PC: Mevalotin 30 minutes þ control procedure
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4. L-NAME þ ischemic PC: L-NAME þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 5. L-NAME þ statin PC: L-NAME þ Mevalotin 30 minutes þ control procedure 6. Glibenclamide þ ischemic PC: glibenclamide þ (5minute occlusion þ 5-minute reperfusion) 3 þ control procedure 7. Glibenclamide þ statin PC: glibenclamide þ Mevalotin 30 minutes þ control procedure 8. 5-HD þ ischemic PC: 5-HD þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 9. 5-HD þ statin PC: 5-HD þ Mevalotin 30 minutes þ control procedure 10. Chelerythrine þ ischemic PC: chelerythrine þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 11. Chelerythrine þ statin PC: chelerythrine þ Mevalotin 30 minutes þ control procedure 12. Genistein þ ischemic PC: genistein þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 13. Genistein þ statin PC: genistein þ Mevalotin 30 minutes þ control procedure 14. Sham group
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2.5. Chemicals All chemicals were purchased from Sigma Co. except those that were particularly mentioned. Mevalotin was obtained from the Laboratory of Sankyo Co., Inc. (Tokyo, Japan). Mevalotin was injected intraperitoneally as a single dose of 1 mg/kg prior to the corresponding experiments. 2.6. Statistical analysis All values are expressed as mean ± the standard error of the mean. Analysis of variance, using NewmaneKeuls post hoc test, was used to determine whether any significant differences existed among groups with regard to the IS and area at risk. A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. Cardioprotection from statin PC We determined the decrease of the IS after statin PC using the triphenyltetrazolium method, as described above. Pretreatment with statin reduces the IS significantly (Fig. 1; 22 ± 4% vs. 53 ± 4%, p < 0.01). The cardioprotective effect provided by statin PC is comparable to that provided by ischemic PC (Fig. 1; 24 ± 4% vs. 53 ± 4%, p < 0.01).
2.3. Determination of IS
3.2. Mechanisms of statin PC
As previously described,5 the IS and the area at risk of ventricular myocardium were measured by planimetry after incubation with 1% triphenyltetrazolium (Sigma Co., St Louis, MO, USA) in phosphate buffer (pH 7.4) and 10% formalin. The volume of the left ventricle, the infarct volume, and the volume of the area at risk were then calculated by multiplying each area by the slice thickness and summing the products. The IS was expressed as a percentage of the area at risk.
To investigate the cellular mechanisms of statin PC, we applied various agents prior to statin PC. The main results are depicted in Figs. 1 and 2. Pretreatment with L-NAME completely blocked the effects of IS reduction provided by statin PC (Fig. 1; 48 ± 4% vs. 22 ± 4%, p < 0.01). Both MitoKATP and sarcolemmal KATP channels can antagonize the cardioprotective effects of statin PC (Fig. 1; glibenclamide: 50 ± 4% vs. 22 ± 4%, p < 0.01; 5-HD: 50 ± 4% vs. 22 ± 4%, p < 0.01). In addition, protein kinase C antagonist (chelerythrine) and tyrosine kinase antagonist (genistein) also have
2.4. Measurement of eNOS messenger RNA by reverse transcriptase-polymerase chain reaction Myocardial tissue samples were obtained from the rats injected with Mevalotin (~1 mg/kg) or saline vehicle and rapidly frozen in liquid nitrogen. Total RNA was extracted from mouse heart, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described previously.35 The mouse eNOS fragment was amplified using the sense primer (50 -GCAGAAGAGTCCAGCGAACA-30 )36 and the antisense primer (50 -GGCAGCCAAACACCAAAGTC-30 ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The mouse GAPDH fragment was amplified using the sense primer (50 -CGGAGTCAACG GATTTGGTCGTAT-30 ) and the antisense primer (50 -AGCCT TCTCCATGGTGGTGAAGAC-30 ). The expression of eNOS messenger RNA (mRNA) was normalized to the level of GAPDH mRNA in each experiment.
Fig. 1. Infarct size/area at risk in various experiment groups (statin PC). 5-HD ¼ 5-hydroxydecanoic acid; L-NAME ¼ NG-nitro-4-arginine methyl ester; PC ¼ preconditioning; SPC ¼ statin preconditioning.
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the same as those observed in the case of statin PC. Activation of myocardial eNOS was observed in the myocardium treated with ischemic PC (1.57 ± 0.2-fold vs. control, p < 0.05). L-NAME can block the statin-induced eNOS mRNA expression completely. Antagonism of sarcolemmal KATP channels, tyrosine kinase C, MitoKATP channels, and protein kinase C restores and even enhances the activation of eNOS mRNA. These results may further confirm that there is a positive feedback in eNOSeNO donationeMitoKATP channels. 4. Discussion
Fig. 2. Infarct size/area at risk in various experiment groups (ischemic PC). 5HD ¼ 5-hydroxydecanoic acid; IPC ¼ ischemic preconditioning; LNAME ¼ NG-nitro-4-arginine methyl ester; PC ¼ preconditioning.
similar effects (Fig. 1; chelerythrine: 48 ± 4% vs. 22 ± 4%, p < 0.01; genisteine: 45 ± 4% vs. 22 ± 4%, p < 0.01). On comparison with Fig. 2, we found that the cellular mechanisms of statin PC are similar to those of ischemic PC. These observations imply that the donation of NO to the downstream KATP channels remains the key pathway involved in myocardial PC. 3.3. Cardiomyocyte eNOS activation by statin PC Activation of myocardial eNOS activation was observed in the myocardium treated with statin PC (1.60 ± 0.2-fold vs. control, p < 0.05). L-NAME can block the statin-induced eNOS mRNA expression completely. However, antagonism of sarcolemmal KATP channels, tyrosine kinase C, MitoKATP channels, and protein kinase C all restore and even enhance the activation of eNOS mRNA (see Fig. 3). These results may imply that eNOSeNO donationeMitoKATP form a positive feedback. To prove these points, we investigated the eNOS mRNA expression by RT-PCR. Surprisingly, the results were almost
Fig. 3. Expression of eNOS mRNA by RT-PCR. 5-HD ¼ 5-hydroxydecanoic acid; eNOS ¼ endothelial nitrous oxide synthase; L-NAME ¼ NG-nitro-4arginine methyl ester; PC ¼ preconditioning; RT-PCR ¼ reverse transcriptase-polymerase chain reaction; SPC ¼ statin preconditioning.
This study demonstrated that preadministration of statin can provoke cardioprotective effects and reduce the IS in a rat myocardial infarction model. Cardioprotective effects are, at least partially, related to the activation of the myocardial eNOS system and KATP channels, which are modified by an additional signaling pathway such as the tyrosine kinase and protein kinase C system. There are positive feedbacks between the tyrosine kinase/protein kinase C system and eNOSeNOeMitoKATP channels. Statins are well known to be associated with NOS activation. NO is a unique intra/intercellular messenger involved in the regulation of many physiological functions such as central and peripheral neurotransmission, smooth muscle contractility, platelet reactivity, and angiogenesis, among others.28 Inappropriate production of NO has been linked to a number of pathologies; therefore, compounds that modulate its release may display a great therapeutic value.29 NO is generated by a family of enzymes known as NOS, named after the tissues in which they were originally described. However, eNOS and neuronal NOS are constitutively expressed not only in endothelia and neurons, but also in many other cell types.30 They release NO in response to receptor stimulation after activation of the Ca2þ/calmodulin-dependent pathway.37 More recently, different kinds of stimuli have been proved to activate eNOS in a Ca2þecalmodulin-independent way. It has been reported that basic fibroblast growth factor (FGF), similar to what was reported for bradykinin,38 is able to activate eNOS in Chinese hamster ovary-k1 cells, via the sphingomyelinase-dependent generation of ceramide, causing translocation of eNOS from the plasma membrane, where it is bound to caveolin-1, to the cytosol in its active form.39 Among the different roles attributed to NO in physiology and pathology, its involvement in carcinogenesis and angiogenesis has achieved great importance recently.40e42 NO has been reported to induce vascular endothelial growth factor expression in carcinoma cells43 and neovascularization in tumors.44 Moreover, angiogenic factors, including both basic FGF and vascular endothelial growth factor, are powerful NOS activators.39,41 Thus, identification of compounds with antiangiogenic properties is of pivotal relevance for the inhibition of tumor growth, and substances able to block NO production may be active in this respect. The tested statins, i.e., lovastatin, atorvastatin, pravastatin, and simvastatin, demonstrate variable potency to enhance the NO/ oxygen concentration ratio after stimulation of NOS, resulting in an increase of NO bioavailability in endothelial cells.37
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It has been proved that early administration of statins can minimize the deteriorating effect of transient cerebral or myocardial ischemia in animal models.31,32 These and other studies elucidate a role for NOS activated by statins.33 IN addition, some statins have been proved to attenuate oxidantinduced mitochondrial dysfunction in cardiac myocytes.34,35 These basic studies supported the clinical observation that statins have certain cardioprotective effects. For example, the incidence of myonecrosis in patients undergoing rotational atherectomy was evaluated by preprocedural statin use. The incidence of any myonecrosis or significant myonecrosis was significantly lower among patients pretreated with statins. This difference remained significant after multivariable and propensity adjustment.35 Our study has certain limitations. The first is that there is a doseeeffect relationship between the glibenclamide dose and its effect on ischemic PC and statin PC, as evident from our data. As mentioned previously, the doses of glibenclamide that completely blocked PC varied widely.3,12,45e48 The differences may arise from the use of different animals for various experiments. In most of the previous studies the Langendorff isolated heart preparation was utilized, whereas we used intact animals. In vivo pharmacokinetics may be complicated and even individualized. However, we believe that such a model can mimic actual pathophysiological conditions in clinical settings. The second limitation is that we cannot demonstrate whether the cellular mechanisms of statin PC are different among animals of different age groups. The results of this study are similar to those of previous studies in which younger animals were used for experiments. Further study designs comparing the degree of cardioprotective effects of statin and the differences in possibly involving signaling pathways may be needed. In this study, we have focused upon the acute cardioprotective effects of statin PC. It remains to be answered whether such protective effects persist or whether delayed statin PC can occur. In summary, Mevalotin, similar to other statins, has been proved to provide cardioprotective effects mimicking ischemic PC. Activated myocardial eNOS system and KATP channels are the main signaling pathways. Conflict of interest The author has no conflicts of interest to declare. Acknowledgments This study was supported by a grant from Shin-Kong Wu Ho-Su Memorial Hospital (SKH-8302-96-DR-28). References 1. Wang TL, Chang H, Hung CR, Tseng YZ. Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction. Cardiovasc Res. 1998;40:557e563.
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2. Wang TL, Chang H, Hung CR, Tseng YZ. Attenuation of neutrophil and endothelial activation by intravenous morphine in patients with acute myocardial infarction. Am J Cardiol. 1997;80:1532e1535. 3. Wang TL, Huang YH, Chang H. Somatostatin analogue mimics acute ischemic preconditioning in a rat model of myocardial infarction. J Cardiovasc Pharmacol. 2005;45:327e332. 4. Banerjee A, Locke Winter C, Rogers KB, et al. Preconditioning against myocardial dysfunction after ischemia and reperfusion by an alpha 1-adrenergic mechanism. Circ Res. 1993;73:656e670. 5. Hoshida S, Kuzuya T, Nishida M, et al. Adenosine blockade during reperfusion reverses the infarct limiting effect in preconditioned canine hearts. Cardiovasc Res. 1994;28:1083e1088. 6. Schulz R, Rose J, Heusch G. Involvement of activation of ATP-dependent potassium channels in ischemic preconditioning in swine. Am J Physiol. 1994;267:H1341eH1352. 7. Marber MS, Latchman DS, Walker JM, Yellon DM. Cardiac stress protein elevation 24 h after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation. 1993;88:1264e1272. 8. Tritto I, D'Andrea D, Eramo N, et al. Oxygen radicals can induce preconditioning in rabbit hearts. Circ Res. 1997;80:743e748. 9. Zahler S, Kupatt C, Becker BF. Endothelial preconditioning by transient oxidative stress reduces inflammatory responses of cultured endothelial cells to TNF-a. FASEB J. 2000;14:555e564. 10. Zhao ZQ, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol. 2003;285:H579eH588. 11. Zahler S, Kupatt C, Mobert J, et al. Effects of ACE-inhibition on redox status and expression of P-selectin of endothelial cells subjected to oxidative stress. J Mol Cell Cardiol. 1997;29:2953e2960. 12. O'Rourke B. Myocardial KATP channels in preconditioning. Circ Res. 2000;87:845e855. 13. Halestrap AP. The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism. Biochim Biophys Acta. 1989;973:355e382. 14. Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive Kþ channel openers prevent Ca2þ overload in rat cardiac mitochondria. J Physiol (Lond). 1999;519:347e360. 15. Vanden Hoek TL, Becker LB, Shao Z, Li C, Schumacker PT. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem. 1998;273:18092e18098. 16. Das DK, Engelman RM, Maulik N. Oxygen free radical signaling in ischemic preconditioning. Ann N Y Acad Sci. 1999;874:49e65. 17. Chen W, Gabel S, Steenbergen C, Murphy E. A redox-based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res. 1995;77:424e429. 18. Zhou X, Ahai X, Ashraf M. Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes. Circulation. 1996;93:1177e1184. 19. Jerome SN, Akimitsu T, Gute DC, Korthuis RJ. Ischemic preconditioning attenuates capillary no-reflow induced by prolonged ischemia and reperfusion. Am J Physiol. 1995;268:H2063eH2067. 20. Defily DV, Chilian WM. Preconditioning protects coronary arteriolar endothelium from ischemiaereperfusion injury. Am J Physiol. 1995;268: H700eH706. 21. Rischard V, Kaeffer N, Tron C, Thuillez C. Ischemia preconditioning protects against coronary endothelial dysfunction induced by ischemia and reperfusion. Circulation. 1994;89:1254e1261. 22. Sphepherd J, Cobbe SM, Ford I, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med. 1995;333:1301e1307. 23. Sacks FM, Pfeffer MA, Moye LA, et al. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996;355:1001e1009. 24. Pitt B, Waters D, Brown WV, et al. Aggressive lipid-lowering therapy compared with angioplasty in stable coronary artery disease. N Engl J Med. 1999;341:70e76.
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25. Takemoto M, Liao JK. Pleiotropic effects of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors. Arterioscler Thromb Vasc Biol. 2001;21:1712e1719. 26. Bell RM, Yellon DM. Atorvastatin, administered at the onset of reperfusion, and independent of lipid lowering, protects the myocardium by up-regulating a pro-survival signaling. Circ Res. 2001;89:1191e1198. 27. Efthymiou CA, Mocanu MM, Yellon DM. Atorvastatin and myocardial reperfusion: new pleiotropic effect implicating multiple prosurvival signaling. J Cardiovasc Pharmacol. 2005;45:247e252. 28. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994;78:915e918. 29. Hobbs AJ, Higgs A, Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol. 1999;39: 191e220. 30. Dobrucki LW, Kalinowski L, Dobrucki IT, Malinski T. Statin-stimulated nitric oxide release from endothelium. Med Sci Monit. 2001;7:622e627. 31. Endres M, Laufs U, Huang Z, et al. Stroke protection by 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998;95:8880e8885. 32. Jones SP, Lefer DJ. Cardioprotective actions of acute HMG-CoA reductase inhibition in the setting of myocardial infarction. Acta Physiol Scand. 2001;173:139e143. 33. Laufs U, Fata VL, Liao JK. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase blocks hypoxia-mediated downregulation of endothelial nitric oxide synthase. J Biol Chem. 1996;272: 31725e31729. 34. Jones SP, Teshima Y, Alao M, Marban E. Simvastatin attenuates oxidantinduced mitochondrial dysfunction in cardiac myocytes. Circ Res. 2003;93:697e699. 35. Gurm HS, Breitbart Y, Vivekanathan D, et al. Preprocedural statin use is associated with a reduced hazard of postprocedural myonecrosis in patients undergoing rotational atherectomy: a propensity-adjusted analysis. Am Heart J. 2006;151:1038.e1e1038.e6. 36. Kawachi S, Hines IN, Laroux FS, et al. Nitric oxide synthase and postischemic liver injury. Biochem Biophys Res Commun. 2000;276:851e854.
37. Igarashi J, Thatte HS, Prabhakar P, Golan DE, Michel T. Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci U S A. 1999;96:12583e12588. 38. Florio T, Arena S, Pattarozzi A, et al. Basic fibroblast growth factor activates endothelial nitric-oxide synthase in CHO-K1 cells via the activation of ceramide synthesis. Mol Pharmacol. 2003;63:297e310. 39. Garcia-Cardena G, Folkman J. Is there a role for nitric oxide in tumor angiogenesis? J Natl Cancer Inst. 1998;90:560e561. 40. Ziche M, Morbidelli L, Choudhuri R, et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest. 1997;99: 2625e2634. 41. Gallo O, Masini E, Morbidelli L, et al. Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer. J Natl Cancer Inst. 1998;90:587e596. 42. Chin K, Kurashima Y, Ogura T, Tajiri H, Yoshida S, Esumi H. Induction of vascular endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells. Oncogene. 1997;15: 437e442. 43. Jenkins DC, Charles IG, Thomsen LL, et al. Roles of nitric oxide in tumor growth. Proc Natl Acad Sci U S A. 1995;92:4392e4396. 44. Sasaki N, Sato T, Ohler A, O'Rourke B, Marban E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation. 2000;101:439e445. 45. Cohen MV, Baines CP, Downey JM. Ischemic preconditioning: from adenosine receptor of KATP channel. Annu Rev Physiol. 2000;62:79e109. 46. Schultz JEJ, Hsu AK, Gross GJ. Morphine mimics cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ Res. 1996;78:1100e1104. 47. Gross GJ, Fryer RM. Sarcolemmal versus mitochondrial ATP-sensitive Kþ channels and myocardial preconditioning. Circ Res. 1999;84: 973e979. 48. O'Rourke B. Evidence for mitochondrial Kþ channels and their role in cardioprotection. Circ Res. 2004;94:420e432.
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Original Research
Statin (Mevalotin) preconditioning decreases infarct size in senile rat myocardial infarction model Tzong-Luen Wang a,b,* a
Department of Emergency Medicine, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b Medical School, Fu-Jen Catholic University, Taipei, Taiwan Received 15 July 2014; accepted 28 July 2014 Available online 18 September 2014
Abstract Aim: Although statins have been proved to have cardioprotective effects, it remains to be investigated whether “statin-induced preconditioning (PC)” can reduce the infarct size for senile animals. Methods: Senile Wistar rats (weighing 350e450 g) were randomly assigned to one of 14 experiment groups (n 5 for each group). All hearts underwent 30 minutes of coronary artery occlusion and 2 hours of reperfusion (control procedure). Ischemic PC was elicited by three cycles of 5-minute occlusion and 5-minute reperfusion. Statin PC was performed by administering Mevalotin (1 mg/kg, the optimal dose being determined by a doseeeffect study) 30 minutes prior to the control procedure. In addition, pretreatment with glibenclamide, 5-hydroxydecanoic acid, chelerythrine, genistein, and NG-nitro-4-arginine methyl ester, or their combination, was also performed to test possible signaling mechanisms. Results: Statin PC decreased the infarct size significantly compared with the control group (22 ± 4% vs. 53 ± 4%, p < 0.01). Pretreatment with NG-nitro-4-arginine methyl ester completely blocked the effects of Mevalotin (infarct size: 48 ± 4% vs. 22 ± 4%, p < 0.01). The cardioprotective effects of statin PC could be blunted by antagonism of mitochondrial adenosine triphosphate (ATP)-sensitive potassium and sarcolemmal ATP-sensitive potassium channels (glibenclamide: 50 ± 4% vs. 22 ± 4%, p < 0.01; 5-hydroxydecanoic acid: 50 ± 4% vs. 22 ± 4%, p < 0.01). In addition, protein kinase C antagonist (chelerythrine) and tyrosine kinase antagonist (genisteine) also have similar effects (chelerythrine: 48 ± 4% vs. 22 ± 4%, p < 0.01; genistein: 45 ± 4% vs. 22 ± 4%, p < 0.01). Conclusion: It is established in this study the myocardial PC caused by statins, that is involved cellular mechanisms including mainly the pathway of endothelial nitrous oxide synthase (eNOS)enitrous oxide donationeATP-sensitive potassium channels. Protein kinase C and tyrosine kinase have regulatory effects. Copyright © 2014, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: endothelial nitrous oxide synthase; mitochondrial adenosine triphosphate-sensitive potassium; preconditioning; statins
1. Introduction Preconditioning (PC) involves a pretreatment or premaneuver that is able to decrease myocardial damage after a severe decrease in coronary flow. We have demonstrated many modes of PC in previous experiments to show cardioprotective effects.1e3 The term “preconditioning” means a powerful * Department of Emergency Medicine, Shin-Kong Wu Ho-Su Memorial Hospital, 95 Wen Chang Road, Taipei, Taiwan. E-mail address: [email protected].
experimental tool that can reduce the infarct size (IS) after ischemiaereperfusion or other pretreatments such as adrenergic stimulation,4 activation of adenosine receptors5 and adenosine triphosphate (ATP)-sensitive potassium (KATP) channels,6 and induction of heat shock proteins7 and oxidative stress.8 Some investigators have further studied the possible effector cells of such PC. Besides neutrophils, which we earlier proposed to be affected by PC,1 endothelial cells are the main target cells at present.2,9e11 Activation of G protein-linked phospholipase C, tyrosine kinase pathways, and protein kinase C, and generation of
http://dx.doi.org/10.1016/j.jacme.2014.07.001 2211-5587/Copyright © 2014, Taiwan Society of Emergency Medicine. Published by Elsevier Taiwan LLC. All rights reserved.
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oxygen radicals are usually involved in PC.12 In addition, KATP channels are considered the most possible triggers and effectors of PC.12 Recent studies suggested that mitochondrial KATP channels (MitoKATP channels), in addition to sarcolemmal KATP channels, might account mainly for the protective mechanism.12e16 MitoKATP channel opening has been proved to be related to mitochondrial swelling and optimization of respiration,13 prevention of mitochondrial calcium overload,14 and control of reactive oxygen radicals,15,16 all of which are possibly linked to cardioprotection during severe myocardial ischemia and infarction. As mentioned above, cellular targets of PC seem to be heterogeneous. Although saline-perfused isolated hearts or isolated cardiomyocytes can be preconditioned,17,18 the socalled microvascular component of PC has been presented.19e21 The concept is concerned with the reduction of postischemic inflammation or mitigation of leukocyte adhesion in the perfused vascular bed after PC. Endothelial cells, the interface between parenchymal tissue and blood cells, are involved in the significant pathophysiological process. On the other hand, statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been available for decades. Some clinical trials have indicated that statins appear to have beneficial effects independent of their cholesterollowering properties, when compared with other agents. These so-called pleiotropic properties include antiinflammatory effects, plaque stabilization,22e24 improved endothelial function, and inhibition of vascular smooth muscle cell proliferation.25 The beneficial pleiotropic effects of statins in protecting the isolated murine hearts against lethal reperfusion injury via activation of phosphatidylinositide 3-kinases (PI3K)/Akt prosurvival signaling pathway and its downstream effectors, endothelial nitrous oxide synthase (eNOS), were also demonstrated.26 In addition, phosphorylation of multiple prosurvival pathways involving p44/p42 Mitogen-activated protein kinases (MAPK), p38 MAPK, and heat shock protein (HSP) 27 were also demonstrated in a later study.27 It is well known that statins are associated with nitrous oxide synthase (NOS) activation. Nitrous oxide (NO) is a unique intra/intercellular messenger involved in the regulation of many physiological functions.28 Any compounds that modulate NO release may display a great therapeutic value.29 Different statins such as lovastatin, atorvastatin, pravastatin, and simvastatin demonstrate variable potency to enhance the NO/oxygen concentration ratio by enhancing NOS, resulting in increased endothelial NO activities.30 In addition, acutely administered statins minimize the damaging effect of transient cerebral31 or myocardial ischemia32 in animal models. Many studies31e33 clearly indicate the role of NOS activated by statins. In addition, some other statins have been proved to attenuate oxidant-induced mitochondrial dysfunction in cardiac myocytes.34 Clinical observations also demonstrated significant protection from myonecrosis among patients undergoing atherectomy with statins.35 We designed the following in vivo study to investigate the presence of the phenomenon “statin-induced PC” and its cellular mechanisms.
2. Methods 2.1. Animal preparation Male Wistar rats, weighing 350e450 g and fed a standard diet, were included in this study. After induction of anesthesia with intraperitoneal pentobarbitone (50 mg/kg body weight), all the animals were ventilated with room air via endotracheal intubation and a rodent respirator (Harvard Apparatus, Boston, MA, USA) at 60e70 breaths per minute. The right carotid artery was cannulated to measure the mean blood pressure and heart rate via a Could PE-50 pressure transducer, which was connected to a Grass polygraph (Grass Technologies, RI. USA). The right jugular vein was cannulated to infuse saline or drugs. A left thoracotomy was performed to expose the heart at the fifth intercostal space. After removing the pericardium, a 6-0-Prolene ligation, together with a snare occluder, was placed over the origin of the left anterior descending artery. Regional ischemia was achieved by pulling the snare and securing the threads with a mosquito hemostat, which indicated that approximately 60% of the myocardium was ischemic in our study. 2.2. Experimental protocols The rats included in the present study were randomly assigned to one of the experimental groups (n 5 for each group). All hearts underwent 30 minutes of coronary artery occlusion and 2 hours of reperfusion (control procedure) after an initial 30-minute stabilization period. Ischemic PC was elicited by three cycles of 5-minute occlusion and 5-minute reperfusion. Statin PC was performed by administering the drug Mevalotin (1 mg/kg, the optimal dose being determined by a doseeeffect study) 30 minutes prior to the control procedure. Glibenclamide (2 mg/kg), a sarcolemmal KATP channel antagonist, was administered 30 minutes prior to statin PC to examine the role of the channel. The effect of 5-hydroxydecanoic acid (5-HD), a MitoKATP channel-selective antagonist, was also examined by giving 10 mg/kg 5-HD 5 minutes prior to the statin PC. To evaluate the possible roles of protein kinase C and tyrosine kinase, their antagonists, chelerythrine (2 mg/kg, 30 minutes prior to statin PC) and genistein (5 mg/kg, 5 minutes prior to statin PC), or their combination for synergic effects, were also given as a pretreatment in various groups. To test the role of NOS in statin-induced cardioprotection, we also determined the effects of the NOS inhibitor NG-nitro-4-arginine methyl ester (L-NAME). L-NAME (50 mg/kg) was administered 30 minutes prior to ischemic PC with or without statin pretreatment, and also prior to statin PC. In summary, the rats were assigned to the following groups for carrying out the experiments: 1. Control group: 30-minute occlusion þ 2-hour reperfusion (control procedure) 2. Ischemic PC: (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 3. Statin PC: Mevalotin 30 minutes þ control procedure
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4. L-NAME þ ischemic PC: L-NAME þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 5. L-NAME þ statin PC: L-NAME þ Mevalotin 30 minutes þ control procedure 6. Glibenclamide þ ischemic PC: glibenclamide þ (5minute occlusion þ 5-minute reperfusion) 3 þ control procedure 7. Glibenclamide þ statin PC: glibenclamide þ Mevalotin 30 minutes þ control procedure 8. 5-HD þ ischemic PC: 5-HD þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 9. 5-HD þ statin PC: 5-HD þ Mevalotin 30 minutes þ control procedure 10. Chelerythrine þ ischemic PC: chelerythrine þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 11. Chelerythrine þ statin PC: chelerythrine þ Mevalotin 30 minutes þ control procedure 12. Genistein þ ischemic PC: genistein þ (5-minute occlusion þ 5-minute reperfusion) 3 þ control procedure 13. Genistein þ statin PC: genistein þ Mevalotin 30 minutes þ control procedure 14. Sham group
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2.5. Chemicals All chemicals were purchased from Sigma Co. except those that were particularly mentioned. Mevalotin was obtained from the Laboratory of Sankyo Co., Inc. (Tokyo, Japan). Mevalotin was injected intraperitoneally as a single dose of 1 mg/kg prior to the corresponding experiments. 2.6. Statistical analysis All values are expressed as mean ± the standard error of the mean. Analysis of variance, using NewmaneKeuls post hoc test, was used to determine whether any significant differences existed among groups with regard to the IS and area at risk. A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. Cardioprotection from statin PC We determined the decrease of the IS after statin PC using the triphenyltetrazolium method, as described above. Pretreatment with statin reduces the IS significantly (Fig. 1; 22 ± 4% vs. 53 ± 4%, p < 0.01). The cardioprotective effect provided by statin PC is comparable to that provided by ischemic PC (Fig. 1; 24 ± 4% vs. 53 ± 4%, p < 0.01).
2.3. Determination of IS
3.2. Mechanisms of statin PC
As previously described,5 the IS and the area at risk of ventricular myocardium were measured by planimetry after incubation with 1% triphenyltetrazolium (Sigma Co., St Louis, MO, USA) in phosphate buffer (pH 7.4) and 10% formalin. The volume of the left ventricle, the infarct volume, and the volume of the area at risk were then calculated by multiplying each area by the slice thickness and summing the products. The IS was expressed as a percentage of the area at risk.
To investigate the cellular mechanisms of statin PC, we applied various agents prior to statin PC. The main results are depicted in Figs. 1 and 2. Pretreatment with L-NAME completely blocked the effects of IS reduction provided by statin PC (Fig. 1; 48 ± 4% vs. 22 ± 4%, p < 0.01). Both MitoKATP and sarcolemmal KATP channels can antagonize the cardioprotective effects of statin PC (Fig. 1; glibenclamide: 50 ± 4% vs. 22 ± 4%, p < 0.01; 5-HD: 50 ± 4% vs. 22 ± 4%, p < 0.01). In addition, protein kinase C antagonist (chelerythrine) and tyrosine kinase antagonist (genistein) also have
2.4. Measurement of eNOS messenger RNA by reverse transcriptase-polymerase chain reaction Myocardial tissue samples were obtained from the rats injected with Mevalotin (~1 mg/kg) or saline vehicle and rapidly frozen in liquid nitrogen. Total RNA was extracted from mouse heart, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described previously.35 The mouse eNOS fragment was amplified using the sense primer (50 -GCAGAAGAGTCCAGCGAACA-30 )36 and the antisense primer (50 -GGCAGCCAAACACCAAAGTC-30 ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The mouse GAPDH fragment was amplified using the sense primer (50 -CGGAGTCAACG GATTTGGTCGTAT-30 ) and the antisense primer (50 -AGCCT TCTCCATGGTGGTGAAGAC-30 ). The expression of eNOS messenger RNA (mRNA) was normalized to the level of GAPDH mRNA in each experiment.
Fig. 1. Infarct size/area at risk in various experiment groups (statin PC). 5-HD ¼ 5-hydroxydecanoic acid; L-NAME ¼ NG-nitro-4-arginine methyl ester; PC ¼ preconditioning; SPC ¼ statin preconditioning.
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the same as those observed in the case of statin PC. Activation of myocardial eNOS was observed in the myocardium treated with ischemic PC (1.57 ± 0.2-fold vs. control, p < 0.05). L-NAME can block the statin-induced eNOS mRNA expression completely. Antagonism of sarcolemmal KATP channels, tyrosine kinase C, MitoKATP channels, and protein kinase C restores and even enhances the activation of eNOS mRNA. These results may further confirm that there is a positive feedback in eNOSeNO donationeMitoKATP channels. 4. Discussion
Fig. 2. Infarct size/area at risk in various experiment groups (ischemic PC). 5HD ¼ 5-hydroxydecanoic acid; IPC ¼ ischemic preconditioning; LNAME ¼ NG-nitro-4-arginine methyl ester; PC ¼ preconditioning.
similar effects (Fig. 1; chelerythrine: 48 ± 4% vs. 22 ± 4%, p < 0.01; genisteine: 45 ± 4% vs. 22 ± 4%, p < 0.01). On comparison with Fig. 2, we found that the cellular mechanisms of statin PC are similar to those of ischemic PC. These observations imply that the donation of NO to the downstream KATP channels remains the key pathway involved in myocardial PC. 3.3. Cardiomyocyte eNOS activation by statin PC Activation of myocardial eNOS activation was observed in the myocardium treated with statin PC (1.60 ± 0.2-fold vs. control, p < 0.05). L-NAME can block the statin-induced eNOS mRNA expression completely. However, antagonism of sarcolemmal KATP channels, tyrosine kinase C, MitoKATP channels, and protein kinase C all restore and even enhance the activation of eNOS mRNA (see Fig. 3). These results may imply that eNOSeNO donationeMitoKATP form a positive feedback. To prove these points, we investigated the eNOS mRNA expression by RT-PCR. Surprisingly, the results were almost
Fig. 3. Expression of eNOS mRNA by RT-PCR. 5-HD ¼ 5-hydroxydecanoic acid; eNOS ¼ endothelial nitrous oxide synthase; L-NAME ¼ NG-nitro-4arginine methyl ester; PC ¼ preconditioning; RT-PCR ¼ reverse transcriptase-polymerase chain reaction; SPC ¼ statin preconditioning.
This study demonstrated that preadministration of statin can provoke cardioprotective effects and reduce the IS in a rat myocardial infarction model. Cardioprotective effects are, at least partially, related to the activation of the myocardial eNOS system and KATP channels, which are modified by an additional signaling pathway such as the tyrosine kinase and protein kinase C system. There are positive feedbacks between the tyrosine kinase/protein kinase C system and eNOSeNOeMitoKATP channels. Statins are well known to be associated with NOS activation. NO is a unique intra/intercellular messenger involved in the regulation of many physiological functions such as central and peripheral neurotransmission, smooth muscle contractility, platelet reactivity, and angiogenesis, among others.28 Inappropriate production of NO has been linked to a number of pathologies; therefore, compounds that modulate its release may display a great therapeutic value.29 NO is generated by a family of enzymes known as NOS, named after the tissues in which they were originally described. However, eNOS and neuronal NOS are constitutively expressed not only in endothelia and neurons, but also in many other cell types.30 They release NO in response to receptor stimulation after activation of the Ca2þ/calmodulin-dependent pathway.37 More recently, different kinds of stimuli have been proved to activate eNOS in a Ca2þecalmodulin-independent way. It has been reported that basic fibroblast growth factor (FGF), similar to what was reported for bradykinin,38 is able to activate eNOS in Chinese hamster ovary-k1 cells, via the sphingomyelinase-dependent generation of ceramide, causing translocation of eNOS from the plasma membrane, where it is bound to caveolin-1, to the cytosol in its active form.39 Among the different roles attributed to NO in physiology and pathology, its involvement in carcinogenesis and angiogenesis has achieved great importance recently.40e42 NO has been reported to induce vascular endothelial growth factor expression in carcinoma cells43 and neovascularization in tumors.44 Moreover, angiogenic factors, including both basic FGF and vascular endothelial growth factor, are powerful NOS activators.39,41 Thus, identification of compounds with antiangiogenic properties is of pivotal relevance for the inhibition of tumor growth, and substances able to block NO production may be active in this respect. The tested statins, i.e., lovastatin, atorvastatin, pravastatin, and simvastatin, demonstrate variable potency to enhance the NO/ oxygen concentration ratio after stimulation of NOS, resulting in an increase of NO bioavailability in endothelial cells.37
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It has been proved that early administration of statins can minimize the deteriorating effect of transient cerebral or myocardial ischemia in animal models.31,32 These and other studies elucidate a role for NOS activated by statins.33 IN addition, some statins have been proved to attenuate oxidantinduced mitochondrial dysfunction in cardiac myocytes.34,35 These basic studies supported the clinical observation that statins have certain cardioprotective effects. For example, the incidence of myonecrosis in patients undergoing rotational atherectomy was evaluated by preprocedural statin use. The incidence of any myonecrosis or significant myonecrosis was significantly lower among patients pretreated with statins. This difference remained significant after multivariable and propensity adjustment.35 Our study has certain limitations. The first is that there is a doseeeffect relationship between the glibenclamide dose and its effect on ischemic PC and statin PC, as evident from our data. As mentioned previously, the doses of glibenclamide that completely blocked PC varied widely.3,12,45e48 The differences may arise from the use of different animals for various experiments. In most of the previous studies the Langendorff isolated heart preparation was utilized, whereas we used intact animals. In vivo pharmacokinetics may be complicated and even individualized. However, we believe that such a model can mimic actual pathophysiological conditions in clinical settings. The second limitation is that we cannot demonstrate whether the cellular mechanisms of statin PC are different among animals of different age groups. The results of this study are similar to those of previous studies in which younger animals were used for experiments. Further study designs comparing the degree of cardioprotective effects of statin and the differences in possibly involving signaling pathways may be needed. In this study, we have focused upon the acute cardioprotective effects of statin PC. It remains to be answered whether such protective effects persist or whether delayed statin PC can occur. In summary, Mevalotin, similar to other statins, has been proved to provide cardioprotective effects mimicking ischemic PC. Activated myocardial eNOS system and KATP channels are the main signaling pathways. Conflict of interest The author has no conflicts of interest to declare. Acknowledgments This study was supported by a grant from Shin-Kong Wu Ho-Su Memorial Hospital (SKH-8302-96-DR-28). References 1. Wang TL, Chang H, Hung CR, Tseng YZ. Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction. Cardiovasc Res. 1998;40:557e563.
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