Published online before print September 25, 2003, doi: 10.1161/01.RES.0000097262.21507.DF
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© 2003 American Heart Association, Inc.
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Simvastatin Attenuates Oxidant-Induced Mitochondrial Dysfunction in Cardiac Myocytes
From the Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Eduardo Marbán, MD, PhD, Chief of Cardiology, Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave–Ross 844, Baltimore, MD 21205. E-mail marban{at}jhmi.edu
Abstract
3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors (statins) can exert beneficial effects independently of serum cholesterol reduction by increasing the bioavailability of nitric oxide. However, it is unclear whether statins can exert such effects directly on cardiac myocytes and whether mitochondria are potential targets. Neonatal rat cardiac myocytes were cultured and subjected to oxidant stress (1 hour of 100 µmol/L H2O2). Mitochondrial membrane potential, a key determinant of cardiomyocyte viability, was assessed by flow cytometric analysis of tetramethylrhodamine ethyl ester (TMRE)–loaded cells. Hydrogen peroxide significantly reduced mitochondrial membrane potential. Incubation of the cardiac myocytes in simvastatin (
1 µmol/L) 1 hour before peroxide exposure significantly attenuated the loss of TMRE fluorescence. This effect was inhibited by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or the ATP-sensitive mitochondrial potassium channel (mitoKATP) blocker 5-hydroxydecanoate. Simvastatin attenuates mitochondrial membrane depolarization after exposure to oxidant stress. These findings provide primary evidence that myocytes can act as triggers and effectors in the cardioprotective cascade of simvastatin therapy. These results bear implications of statin therapy as a potential clinical application of pharmacological preconditioning.
Key Words: mitochondria • statins • myocytes • nitric oxide • preconditioning
3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors (statins) have emerged as the most widely used agents to reduce serum cholesterol levels and subsequent risk for coronary events.1 Recent evidence indicates that the beneficial results of statins may not be limited to cholesterol reduction.2 Specifically, acutely administered statins minimize the damaging effect of transient cerebral3 or myocardial ischemia4 in animal models. These and other studies5 clearly indicate a role for nitric oxide synthase (NOS) activation by statins. Although it is generally believed that statins exert these pleiotropic effects through enhanced bioavailability of vascular NOS-derived NO, the in vivo studies were limited by the inability to determine the precise cell type(s) responsible for statin-induced NO production. Furthermore, potential intracellular targets remain unexplored. In the present study, we determined whether simvastatin could exert direct cytoprotective effects in cardiac myocytes independently of other cell types using isolated cardiac myocytes subjected to oxidative stress. An additional aim of this study was to evaluate whether statins could exert protective effects via mitochondrial ATP-sensitive potassium channels (mitoKATP).6
Materials and Methods
Rat Cardiac Myocyte Cultures
Cardiac myocytes were isolated from 1- or 2-day-old Sprague-Dawley rats (Zivic Laboratories, Inc, Pittsburgh, Pa) and cultured in modified DMEM media as previously described.7 Cardiac myocyte cultures were incubated with indicated concentrations of simvastatin for 1 hour before 0.1 mmol/L hydrogen peroxide challenge. Simvastatin (supplied by Dr D.J. Lefer, Louisiana State University Health Sciences Center, Shreveport, La) was activated via alkaline hydrolysis as previously described.8
Flow Cytometric Analysis
Mitochondrial membrane potential (
m) was assessed by tetramethylrhodamine ethyl ester (TMRE; Molecular Probes) using a flow cytometer as previously described.9 TMRE (100 nmol/L) was loaded for 20 minutes in the dark at 37°C. Cardiac myocytes were subjected to flow cytometry by activation with the 488-nm wavelength. Fluorescence was monitored in the FL-2 channel.
Reagents
All chemicals were purchased from Sigma unless otherwise indicated.
Results and Discussion
Maintenance of mitochondrial membrane potential (m) is necessary for production of energy (ATP) and preservation of cellular homeostasis. We have previously demonstrated that maintenance of m is a critical primary determinant of myocyte survival.10 Figure 1A shows results of flow cytometric analysis in cardiomyocytes loaded with the m indicator TMRE. Consistent with previous work, oxidative stress by H2O2 exposure led to a loss of m as indicated by the decrease in fluorescence of the higher peak (>102). In addition, a significant increase in the number of depolarized/dying cells is shown by the enhanced size of the lowest peak (101). One hour of pretreatment with 5 µmol/L simvastatin significantly attenuated the number of cells that lost high levels of fluorescence, indicating preservation of m (Figure 1B). As shown in Figure 1C, the effectiveness of simvastatin in maintaining m was not evident at lower concentrations of simvastatin. At concentrations greater than 1 µmol/L, simvastatin exerted protective effects that appeared maximal by 5 µmol/L. These data demonstrate that simvastatin preserves mitochondrial membrane potential in a dose-dependent manner in response to oxidative stress.
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It has previously been demonstrated that acute treatment with statins produces cardioprotective effects in vivo in a NO-dependent manner.11–13 We tested the role of NOS in simvastatin-induced cardioprotection by determining the effects of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Figures 2A and 2B show that L-NAME blocks the beneficial effects of simvastatin in a cardiomyocyte-specific system. Previous studies have identified the signal transduction pathway(s) through which acute administration of statins can posttranslationally enhance endothelial nitric oxide synthase (eNOS) activity. Namely, Kureishi et al14 found that simvastatin induced the phosphorylation (activation) of Akt, and subsequently eNOS, within 1 hour of a 1 µmol/L treatment. Considering the timing of the simvastatin treatment in the present study (1 hour), our findings are temporally consistent with such a posttranslational process14 and likely are independent of transcriptional changes.
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Considering prior findings indicating that NO donors can activate mitoKATP channels,15 we questioned whether the protective effects of statins were also mitoKATP-dependent. In Figure 2B, blockade of mitoKATP by 5-hydroxydecanoate (5-HD) abrogated the protective effects of simvastatin. These data indicate that statins may, at least partially, operate through activation of mitoKATP channels in cardiac myocytes. While the actual existence of mitoKATP is not universally accepted,16 the present results link statins to the well-accepted mitochondrial mechanisms of pharmacological preconditioning.17
The use of an isolated cardiac myocyte system demonstrates the potential of simvastatin to exert protective effects directly in cardiac myocytes in the absence of other cell types. This suggests that cardiac myocytes per se can serve as the triggers and effectors of the pleiotropism of statins. This possibility does not exclude a role for the vasculoprotective hypothesis of the cholesterol-independent effects of statins. It is indeed likely that statins exert protective effects both in endothelial cells and in cardiac myocytes, and that interactions between the two cell types may occur in vivo.
The use of isolated neonatal myocytes may limit the application of these findings with respect to the intact adult heart. Challenging myocytes with oxidative stress is unlikely to be a solitary cause of cardiac injury in vivo. The dose of simvastatin presented here is also higher than the therapeutic concentration expected in humans. Accordingly, we performed additional experiments to evaluate whether lower (nmol/L) doses of simvastatin for longer durations (24 hours) would produce similar effects to the high-dose acute administration shown here. Such a dosing regimen was unsuccessful in protecting cardiac myocytes (data not shown). However, it is impossible to make direct dosage comparisons between species and between isolated cells and plasma. Furthermore, the doses of simvastatin used in the present study are similar to those used by others in isolated cell models.14,18,19
Presently, we demonstrate a novel paradigm whereby simvastatin, via NOS and mitoKATP channels, results in cardioprotection in cardiac myocytes. These data provide additional insight into the mechanisms of the protective effects of statins. According to the current paradigm, statins activate NOS and promote the vascular bioavailability of NO. The protective effects of NO on the vasculature are well characterized and widely appreciated. In the intact heart, it is likely that statins are exerting effects on multiple cell types. Here, we provide primary evidence that cardiac myocytes may also be beneficiaries of the protective effects of statins. Previous work supports a scenario in which statins act at the vascular level to prevent the occurrence of ischemia and improve perfusion after ischemia both by preventing thrombotic events and maintaining vasorelaxation. Our findings augment this understanding by demonstrating that myocytes are protected at the mitochondrial level, an idea for which significant evidence exists in vivo.20
The clinical evidence21 for cholesterol-independent effects of statins is extensive and growing. The pleiotropic effects of statins may be largely mediated by NO,2 which can activate mitoKATP channels15 and induce cardioprotection.6 The possibility that statins exert effects similar to preconditioning in the clinical setting is intriguing. This may lead one to question whether potential clinical ramifications of pharmacological preconditioning with statins are currently overlooked, a possibility worthy of further examination.
Acknowledgments
This study was funded by grants from the NIH (R37 HL36957 to Dr Marbán and HLO 7227-27) and a Banyu Fellowship in Cardiovascular Medicine (M.A.). Dr Marbán holds the Michel Mirowski, M.D. Professorship of Cardiology.
Footnotes
This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received June 6, 2003; resubmission received August 12, 2003; accepted September 11, 2003.
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