Author + information
- Received June 18, 2010
- Revision received August 15, 2010
- Accepted September 23, 2010
- Published online February 22, 2011.
- Serge Sicouri, MD⁎ (, )
- Brittany Gianetti, BS,
- Andrew C. Zygmunt, PhD,
- Jonathan M. Cordeiro, PhD and
- Charles Antzelevitch, PhD
- ↵⁎Reprint requests and correspondence:
Dr. Serge Sicouri, Masonic Medical Research Laboratory, 2150 Bleecker Street, Utica, New York 13501-1787
Objectives The purpose of this study was to determine the electrophysiologic effects of simvastatin in canine pulmonary vein (PV) sleeve preparations.
Background Ectopic activity arising from the PV plays a prominent role in the development of atrial fibrillation.
Methods Transmembrane action potentials were recorded from canine superfused left superior or inferior PV sleeves using standard microelectrode techniques. Acetylcholine (1 μM), isoproterenol (1 μM), high calcium ([Ca2+]o = 5.4 mM), or a combination was used to induce early afterdepolarizations or delayed afterdepolarizations and triggered activity. Voltage clamp experiments were performed in the left atrium measuring fast and late sodium currents.
Results Under steady-state conditions, simvastatin (10 nM, n = 9) induced a small increase in action potential duration measured at 85% repolarization and a significant decrease in action potential amplitude, take-off potential, and maximum rate of rise of action potential upstroke at the fastest rates. The Vmax decreased from 175.1 ± 34 V/s to 151.7 ± 28 V/s and from 142 ± 47 V/s to 97.4 ± 39 V/s at basic cycle lengths of 300 and 200 ms, respectively. Simvastatin (10 to 20 nM) eliminated delayed afterdepolarizations and delayed afterdepolarization-induced triggered activity in 7 of 7 PV sleeve preparations and eliminated or reduced late-phase 3 early afterdepolarizations in 6 of 6 PV sleeve preparations. Simvastatin (20 nM) did not affect late or fast sodium currents measured using voltage clamp techniques.
Conclusions Our data suggest that in addition to its upstream actions to reduce atrial structural remodeling, simvastatin exerts a direct antiarrhythmic effect by suppressing triggers responsible for the genesis of atrial fibrillation.
Since their discovery in Japan in the mid-1970s, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) have been widely used to reduce low-density lipoprotein (LDL) and total cholesterol. Other beneficial cardiovascular effects, in addition to those involving the lipid profile, have been attributed to statins. These so-called pleiotropic effects are mainly related to the anti-inflammatory and potential antiarrhythmic effects of statins, particularly in the setting of atrial fibrillation (AF) (1). Experimental studies have shown that statins attenuate atrial electrical remodeling induced by atrial tachypacing and can thus prevent the creation of the substrate for development of AF (2). Simvastatin was shown to greatly attenuate the effect of tachypacing to down-regulate the L-type Ca2+ channel α-subunit (Cav1.2) expression. A number of randomized and large observational clinical studies have reported beneficial effects of statins in AF, particularly in the setting of heart failure. Treatment with statins was linked to a decreased incidence or recurrence of AF in patients with a history of AF, patients undergoing cardiac surgery, post-ablation, patients implanted with a pacemaker or implantable cardioverter-defibrillator, or in patients with acute coronary syndrome (3–7). To the best of our knowledge, no controlled clinical studies have been performed evaluating the acute effect of simvastatin on atrial fibrillation.
Ectopic activity arising from the pulmonary veins (PV) has been shown to play an important role in the development of AF (8). Late-phase 3 early afterdepolarization (EAD)-induced as well as delayed afterdepolarization (DAD)-induced triggered activity originating from PV sleeves have been proposed as potential triggers in the initiation of AF (9–15).
The present study was designed to test the hypothesis that simvastatin's ameliorative effects on AF are in part mediated by its actions to suppress triggered activity arising from PV sleeves.
This investigation conforms to the “Guide for Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication no. 85-23, revised 1996) and was approved by the Animal Care and Use Committee of the Masonic Medical Research Laboratory.
Adult mongrel dogs, weighing 20 to 35 kg, were anticoagulated with heparin (180 IU/kg) and anesthetized with sodium pentobarbital (35 mg/kg, intravenously). The chest was opened through a left thoracotomy, and the heart excised and placed in a cold cardioplegic solution ([K+]0 = 8 mmol/l, 4oC).
Superfused PV sleeve preparation
The PV sleeve preparations (approximately 2.0 × 1.5 cm) were isolated from left canine atria. The canine PV sleeve model consists of a small rectangular section of cardiac muscle, approximately 2 cm by 1.5 cm and 1-mm to 2-mm thick. The preparation is isolated from the left atrium adjacent to the PV. The vein is cut open so that muscle and vein lay flat, thus exposing the surface area from which to record action potentials under control conditions and after exposure to the different solutions used in the study. The tissue is superfused with normal Tyrode solution or Tyrode solution containing simvastatin, isoproterenol, high calcium, or their combinations. Therefore, the different drugs diluted in the Tyrode solution are not directly perfused through the pulmonary vein and should not experience mechanical interference through competitive flow.
Similar to the findings in the human heart (16), histological studies performed in the canine heart have shown that sleeves of endocardium penetrate the proximal section of the PV (17,18). In this study, action potentials were recorded from the endocardial extension of atrial muscle covering the PVs. Left superior PVs were used preferentially in most experiments. The preparations were placed in a small tissue bath and superfused with Tyrode's solution of the following composition (in mM): 129 NaCl, 4 KCl, 0.9 NaH2PO4, 20 NaHCO3, 1.8 CaCl2, 0.5 MgSO4, and 5.5 glucose, buffered with 95% O2/5% CO2 (35 ± 0.5oC). The PV preparations were stimulated at a basic cycle length (BCL) of 1,000 ms during the equilibration period (1 h) using electrical stimulation (1 to 3 ms duration, 2.5 times diastolic threshold intensity) delivered through silver bipolar electrodes insulated except at the tips. Transmembrane potentials were recorded using glass microelectrodes filled with 3 M KCl (10 to 20 MΩ DC resistance) connected to a high input-impedance amplification system (World Precision Instruments, model KS-700, New Haven, Connecticut). The following parameters were measured: take-off potential (TOP), action potential amplitude (APA), action potential duration at 85% repolarization (APD85), and maximal rate of rise of action potential upstroke (Vmax). Transmembrane action potentials were recorded at a sampling rate of 41 kHz. Measurement of TOP was used instead of resting membrane potential because the slow phase 3 of the action potential of the PV sleeve preparation fails to return to the resting potential at the fastest rates.
Isolated myocyte preparation
Atrial myocytes were prepared from canine hearts using techniques described previously (19). Briefly, male and female adult mongrel dogs were anesthetized with sodium pentobarbital (35 mg/kg intravenously), and their hearts were rapidly removed and placed in nominally Ca2+-free Tyrode's solution. The left atria were excised, cannulated through the ostium and perfused with nominally Ca2+-free Tyrode's solution containing 0.1% bovine serum albumin (BSA) for a period of about 5 min. The atria was then subjected to enzyme digestion with the nominally Ca2+-free solution supplemented with 0.5 mg/ml collagenase (Type II, Worthington Biochemicals, Lakewood, New Jersey) and 1 mg/ml BSA for 30 min. After perfusion, tissue from the appendage and the pectinate muscle were isolated, placed in separate beakers, minced and incubated in fresh buffer containing 0.5 mg/ml collagenase, 1 mg/ml BSA and agitated. The supernatant was filtered, centrifuged and the pellet containing the myocytes was stored at room temperature.
Voltage clamp recordings of peak sodium current (INa)
Early INa was measured as previously described (19) with minor modifications. Experiments were performed using a MultiClamp 700A (AXON Instruments, Foster City, California). Command voltages were delivered and data acquired using a DigiData 1322 computer interface using the pClamp 9 program suite (AXON Instruments) with data stored on computer hard disk. Patch pipettes were pulled from borosilicate glass (1.5 mm outer diameter and 1.1 mm inner diameter) on a Model PP-830 vertical puller (Narashige Instruments, Tokyo, Japan). The electrode resistance was 1 to 2.5 MΩ when filled with the internal solution (see the following text). The membrane was ruptured by applying negative pressure and series resistance compensated by 75% to 80%. Whole cell current data was acquired at 20 to 50 kHz and filtered at 5 kHz. Currents were normalized to cell capacitance. All peak INa experiments were performed at 15° C.
External solution contained the following (in mM): NMDG 105, NaCl 40, CaCl2 2.0, MgCl2 1, glucose 10, and HEPES 10, pH adjusted to 7.4 with HCl. The pipette solution contained (mM) NaCl 5, aspartate 120, MgCl2 1, CsCl 5, HEPES 10, MgATP 5, and EGTA 10, pH adjusted to 7.2 with CsOH. Peak sodium current was dramatically reduced in the low extracellular sodium to ensure adequate voltage control. Cells were held at −90 or −120 mV before evoking a train of 15 100-ms pulses to −30 mV with a diastolic interval of 100 ms.
Voltage clamp recordings of late INa
Late INa density was measured in full external Na+ at 37° C as previously described (20). External solution contained (in mM) NaCl 140, CaCl2 2.0, MgCl2 1, glucose 10, and HEPES 10, pH adjusted to 7.4 with NaOH. The pipette solution contained (mM) NaCl 10, aspartate 130, MgCl2 1, CsCl 10, HEPES 10, MgATP 5, and EGTA 10, pH adjusted to 7.2 with CsOH.
Late INa density was recorded in cells that were held at −90 mV. Currents were recorded during a train of 15 pulses. To remove steady-state inactivation, a 160- or 200-ms pulse to −120 mV was taken before a 200-ms pulse to −30 mV. Simvastatin at concentrations of 20 nM or 5 μM was applied and the train repeated, followed by 10 μM tetrodotoxin. Late INa, characterized as the tetrodotoxin-sensitive difference current, was measured as the total charge moved beginning 15 ms after the start of the step to −30 mV.
Whole cell currents were analyzed using the Clampfit analysis program from pClamp 9 suite (AXON Instruments).
Simvastatin was dissolved in dimethyl sulfoxide to form a stock solution of 100 μM and used at a final concentration of 10 to 20 nM. Isoproterenol (Sigma-Aldrich, St. Louis, Missouri) was dissolved in distilled water to form a stock solution of 1 mM and used at final concentrations of 1 and 0.5 μM. It should be noted that a previous study performed in rabbit myocytes (21) used “activated” simvastatin (22) to measure calcium as well as transient outward currents. We used activated simvastatin to measure sodium currents and repeated experiments evaluating the effects of activated simvastatin in PV sleeve preparations. Similar results were observed with activated and nonactivated forms of simvastatin; therefore, the data were pulled together in the results section. Acetylcholine (ACh) was dissolved in distilled water to form a stock solution of 10 mM and used at a concentration of 1 μM.
Electrophysiological data are presented as mean ± SD. Statistical analysis of action potential data obtained in the presence and absence of drug was performed using paired t test, and statistical comparisons of voltage-clamp data were made using analysis of variance followed by a Student Newman-Keuls test. Mean values were considered to be statistically different at p < 0.05.
Effects of simvastatin on PV sleeve preparations
Simvastatin (10 nM) accentuated the depolarization and reduction of take-off potential normally observed in PV sleeve preparations with rapid rates of activation (Fig. 1). The more positive take-off potential at the faster rates is due to a small simvastatin-induced increase in the duration of action potential caused by a slowing of the final part of phase 3. Composite data show a small increase in APD85 and a significant decrease in APA, TOP, and Vmax at the fastest rates (Table 1,Fig. 2). Our findings indicate that simvastatin induces a marked use-dependent depression of Vmax in PV sleeve preparations.
Effects of simvastatin on DAD- and EAD-induced triggered activity
In another series of experiments, we determined the effects of simvastatin on DADs, late-phase 3 EADs, and triggered activity elicited by exposure of the PV sleeve preparations to ACh, isoproterenol, high [Ca2+], or their combination and rapid pacing. Simvastatin (10 to 20 nM) eliminated DADs and DAD-induced triggered activity in 7 of 7 PV sleeve preparations (Fig. 3). Simvastatin (10 to 20 nM) eliminated late phase 3 EADs and triggered activity in 6 of 6 PV sleeve preparations (Fig. 4). Simvastatin also suppressed ACh (1 μM) and high calcium (5.4 mM) induced late phase 3 EADs observed in alternate beats at rapid pacing rates (Fig. 5).
Effects of simvastatin on late and fast sodium currents
Figure 6 shows that INa, late was unaffected by 5 μM simvastatin; Figure 6A shows INa, late in control solutions and 4 min after applying the drug. While exposed to the drug, the diastolic interval was abbreviated from 200 ms to 160 ms, and the currents in Figure 6B were recorded. In the presence of 5 μM simvastatin, INa, late was unaffected by shortening the diastolic interval. Figure 7 summarizes the experiments showing that early and late INa were unaffected by 20 nM simvastatin.
Atrial fibrillation is the most common arrhythmia requiring medical attention. Ectopic activity arising from the PV sleeves is thought to be an important source of triggers and, in some cases, substrate for the development of AF (23–25). The results of the present study indicate that concentrations of simvastatin within the therapeutic range (8 to 20 nM) (17,26) induce rate-dependent depression of Vmax in canine PV sleeve preparations. Our results also indicate that simvastatin suppresses DADs, late-phase 3 EADs, and triggered activity induced in PV sleeves by the addition of isoproterenol, acetylcholine, high calcium, or their combination. These data suggest a direct antiarrhythmic effect of simvastatin, in addition to the previously described upstream therapeutic effect.
Simvastatin eliminates EAD- and DAD-induced triggered activity in PV sleeves
The present study shows that late-phase 3 EADs and DADs can be easily induced in PV sleeve preparations after the addition of isoproterenol, ACh, and high calcium, alone or in combination. Similar findings were previously reported by Chen et al. (17,27) in canine and rabbit isolated single PV myocytes, by Patterson et al. (12,13) in isolated canine PV sleeve preparations, and by Burashnikov et al. (11,28) in coronary-perfused right atrial preparations. In isolated myocytes, isoproterenol was shown to increase automaticity and induce spontaneous as well as EAD- or DAD-induced triggered activity (17,27). Late-phase 3 EADs and late-phase 3 EAD-induced triggered activity (11,28) is a relatively new concept of arrhythmogenesis. Abbreviated repolarization permits normal sarcoplasmic reticulum calcium release and associated sodium-calcium exchange inward current to induce a late-phase 3 EAD, resulting in closely coupled triggered responses when it reaches threshold. Conditions permitting intracellular calcium loading facilitate the development of late-phase 3 EADs. Patterson et al. (12,13) showed that autonomic stimulation could give rise to this phenomenon in canine PV sleeves and in some cases result in a run of triggered responses.
Our data illustrate that simvastatin in concentrations of 10 nM and 20 nM (within its therapeutic range) is able to eliminate late-phase 3 EADs and DAD-induced triggered activity arising from these 2 mechanisms. Similar findings were reported for ranolazine, an antianginal agent with a potent effect to cause use-dependent depression of sodium channel activity at rapid rates, similar to that of simvastatin (9).
In isolated mouse ventricular myocytes, simvastatin has been shown to inhibit the late ICa, although at much higher concentrations (IC50 = 50 μmol/l) (21). The data point to a lack of an effect of simvastatin on either early (peak) and late sodium currents in canine atrial cells despite a significant effect of the drug to reduce Vmax in PV sleeves at fast rates (BCL of 500, 300, and 200 ms). The decrease in Vmax may be explained by a simvastatin-induced depolarization and a more positive take-off potential at the faster rates due to a small simvastatin-induced increase in the duration of action potential caused by a slowing of the final part of phase 3. Additional studies will be necessary to establish the ionic basis for this change. Suppression of DAD and late-phase 3 EAD activities by simvastatin can be explained by its actions to reduce intracellular sodium loading, which occurs at rapid activation rates. By doing so, simvastatin reduces calcium loading, which normally occurs under these conditions as a result of sodium-calcium exchange (29–31). The effects may also have been explained by an effect of simvastatin to block calcium current (21).
Comparison of simvastatin with traditional antiarrhythmic drugs
The antifibrillatory action of most antiarrhythmic agents used to treat AF has been ascribed chiefly to their ability to induce prolongation of the effective refractory period, by prolonging APD (i.e., dl-sotalol and dofetilide), reducing excitability (i.e., flecainide and propafenone), or by a combined mechanism (i.e., amiodarone and ranolazine). In experimental studies, multiple ion-channel blockers, like amiodarone and ranolazine, act by prolonging effective refractory period both by increasing APD as well as by producing a marked depression of excitability, thus inducing post-repolarization refractoriness especially at rapid activation rates (use-dependent effect) (32,33).
Compared to these traditional antiarrhythmic agents, simvastatin produces little change in APD, but does induce a depression of Vmax and excitability at rapid rates, although not as accentuated as in the case of chronic amiodarone (34,35). This action of simvastatin is most similar to that of class IB antiarrhythmic agents such as lidocaine. The effects of simvastatin on EADs and DAD-induced triggered activity are similar to those of ranolazine (9). Chronic amiodarone has also been shown to prevent the development of EADs, DADs, and triggered activity induced by exposure to isoproterenol, ACh, high calcium, or their combination (10,35). Thus, the actions of simvastatin on the triggers of AF (DAD- or EAD-induced triggered activity) are similar to those of traditional antiarrhythmic drugs, whereas its effects on the substrate are less pronounced. These findings suggest that further studies examining the efficacy of a combination of statins with antiarrhythmic agents in the management of AF, may be warranted.
Clinical implications: antiarrhythmic effects of statins
Statins are the most common drugs used in the clinical treatment of hypercholesterolemia. Patients administered statins for their lipid-lowering actions may benefit from their upstream effects (36), which are related to anti-inflammatory actions as well as their direct effects to minimize electrical remodeling (37). Clinical (38) and experimental studies point to a prominent role of PVs in the genesis of atrial arrhythmias, including AF (8,27). Our data suggest that in addition to its upstream effects and direct actions to reduce electrical remodeling during rapid activation of the atria, simvastatin can suppress late-phase 3 EADs, DADs, and triggered activity induced in PV sleeves. Interestingly, other agents with upstream actions, such as the antihypertensive agents losartan and enalapril, have also been shown in preliminary reports to eliminate DADs and late phase 3 EAD-induced triggered activity in PV sleeves (39). Our findings suggest that simvastatin, and perhaps other statins, may exert an important antiarrhythmic effect to suppress the triggers that contribute to the development of AF. Further clinical studies are warranted to evaluate the role of statins in the prevention and treatment of atrial arrhythmias, including AF.
Our data suggest that in addition to its upstream actions to reduce atrial structural remodeling, simvastatin exerts a direct antiarrhythmic effect by suppressing triggers responsible for the genesis of AF.
Supported by grant HL47678 from the National Heart, Lung, and Blood Institute (Dr. Antzelevitch) and New York State and Florida Grand Lodges F. & A.M. All other authors have reported that they have no relationships to disclose.
- Abbreviations and Acronyms
- atrial fibrillation
- action potential duration measured at 85% repolarization
- basic cycle length
- delayed afterdepolarization
- early afterdepolarization
- calcium current
- sodium current
- pulmonary vein
- maximal rate of rise of action potential upstroke
- Received June 18, 2010.
- Revision received August 15, 2010.
- Accepted September 23, 2010.
- American College of Cardiology Foundation
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