- Email: [email protected]
Milrinone inhibits hypoxia or hydrogen dioxide-induced persistent sodium current in ventricular myocytes
European Journal of Pharmacology 616 (2009) 206–212
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Milrinone inhibits hypoxia or hydrogen dioxide-induced persistent sodium current in ventricular myocytes Jie Zheng, Jihua Ma ⁎, Peihua Zhang, Liangkun Hu, Xinrong Fan, Qiong Tang Cardio-Electrophysiological Research Laboratory, Medical College, Wuhan University of Science and Technology, Wuhan, Hubei, 430081, China
a r t i c l e
i n f o
Article history: Received 17 January 2009 Received in revised form 28 May 2009 Accepted 9 June 2009 Available online 21 June 2009 Keywords: Milrinone Persistent sodium current Hypoxia Hydrogen peroxide Patch-clamp
a b s t r a c t Much evidence indicates that increased persistent sodium current (INa.P) is associated with cellular calcium overload and INa.P is considered to be a potential target for therapeutic intervention in ischaemia and heart failure. By inhibiting type III phosphodiesterase, milrinone increases intracellular cyclic adenosine monophosphate (cAMP), with a positive inotropic effect. However, the effect of milrinone on increased INa.P under pathological conditions remains unknown. Accordingly, we investigated the effect of milrinone on increased INa.P induced by hypoxia or hydrogen dioxide in guinea pig ventricular myocytes. While milrinone (0.01 mM or 0.1 mM) or cAMP (0.1 mM) decreased INa.P respectively in control condition, application of 1 μM H-89, a selective cAMP-dependant protein kinase inhibitor, prevented the effect of 0.1 mM milrinone in control condition. Milrinone (0.1 mM) reduced the increased INa.P induced by hypoxia. Furthermore, 0.01 mM or 0.1 mM milrinone reduced the enhanced INa.P induced by 0.3 mM hydrogen peroxide. In addition, 0.01 mM or 0.1 mM milrinone shortened action potential duration at 90% repolarization (APD90). Bath application of 0.3 mM hydrogen dioxide markedly prolonged APD90, while 2 μM tetrodotoxin (TTX) reversed the prolonged APD90. In the other two groups, 0.01 mM or 0.1 mM milrinone shortened the prolonged APD90 induced by 0.3 mM hydrogen peroxide, ultimately 2 μM TTX causing a further decurtation of APD90. These findings demonstrate that milrinone inhibited INa.P under normal condition, hypoxia or hydrogen dioxide-induced INa. P, and the APD90 prolonged by hydrogen dioxide-induced INa.P in ventricular myocytes, which is associated with the mechanism of milrinone increasing intracellular cAMP. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sodium current is an important ion current in cardiac tissues. Under normal condition the amplitude of INaP is much smaller than that of INaT, which is responsible for the upstroke and conduction of the action potential. However, INaP plays an important role in maintaining the plateau of action potential, determining action potential duration (Kiyosue and Arita, 1989), altering [Na+]i (Saint, 2006), transmurally dispersing repolarization and developing cardiac arrhythmias (Ashamalla et al., 2001; Honen and Saint, 2002; Sakmann et al., 2000; Zygmunt et al., 2001). Since Ju et al. (1996) reported that hypoxia could increase persistent sodium current in rat ventricular myocytes, researchers have been paying increasingly attention to INaP. Several studies indicated that a rise of [Na+]i is caused by the increased INaP induced by hypoxia or reactive oxygen species and intracellular calcium overload caused by reactive oxygen species, which results in cell injury or death, results from a rise in [Na+]i followed by Ca2+ influx via the reverse mode of the Na+–Ca2+ exchanger (NCX) (Friedman and Haddad, 1994; Haigney et al., 1994; Song et al., 2006; Wang et al.,
⁎ Corresponding author. Tel./fax: +86 027 68862109. E-mail address: [email protected] (J. Ma). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.06.021
2008). Furthermore, increased INaP causes a prolongation of action potential duration, leading to arrhythmic activity and contractile dysfunction (Maltsev et al., 2007; Song et al., 2006; Wang et al., 2008). Therefore, inhibition of INaP is considered to be a potential target for therapeutic intervention in ischaemia and heart failure (Belardinellia et al., 2004; Hale and Kloner, 2006; Song et al., 2006; Sossalla et al., 2008; Wang et al., 2008). Milrinone represents a second generation of phosphodiesterase inhibitor. By inhibiting Type III phosphodiesterase, milrinone increases intracellular cyclic adenosine monophosphate (cAMP), resulting in a positive inotropic effect on the heart, vasodilatation in the periphery and reduction in ventricular relaxation time (Farah and Frangakis, 1989), without worsening major determinants of myocardial oxygen demand (Niemann et al., 2003). Milrinone is approved for intravenous administration in the treatment of congestive heart failure (Simonton et al., 1985), decompensated congestive heart failure (Shipley et al., 1996) and pulmonary hypertension (Chen et al., 1997). Furthermore, it is used for preventing low cardiac output syndrome after cardiac surgery (Hoffman et al., 2002, 2003). The positive effect of milrinone on cardiac mechanical activity has been reported widely. However, the effect of milrinone on cardiac electrical activity has been seldom studied and its effect on INaP has not been reported at all. While it is widely accepted that milrinone increases
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
intracellular cAMP, our study revealed cAMP can distinctly inhibit INaP. It is significant to investigate the effect of milrinone on INaP for further interpreting pharmacological mechanism of milrinone and expanding the scope of application of milrinone. 2. Materials and methods 2.1. Isolation of ventricular myocytes Adult guinea pigs (250–300 g, of either sex, grade II, Experiment Animal Center of Wuhan University of Science and Technology, Wuhan, China) were anesthetized with pentobarbital sodium (30 mg/kg, i.p.) 20 min after an intraperitoneal injection of 2000 U of heparin. Hearts were excised rapidly and perfused retrogradely on a Langendorff apparatus with a Ca2+-free Tyrode's solution for 5 min before the perfusate was switched to an enzyme-containing solution [0.1 g/l collagenase type I, 0.01 g/l protease E, 0.5 g/l bovine serum albumin (BSA) in the same solution] for 5 min. The perfusate was finally changed to KB solution containing (mM): KOH 70, taurine 20, glutamic acid 50, KCl 40, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, and glucose 10, pH 7.4, for 5 min. These perfusates were bubbled with 100% O2 and maintained at 37 °C. The ventricles were cut into small chunks and gently agitated in KB solution. The cells were filtered through nylon mesh and stored in KB solution at 4 °C. All procedures met the guide for the care and use of laboratory animals regulated by Administrative Regulation of Laboratory Animals of Hubei Province. 2.2. Induction of hypoxia The cell being examined was continuously perfused with extracellular solution flowing through a small plastic tube from a test tube. Hypoxia was achieved by bubbling the perfuse solution (normal Tyrode's solution without glucose) in this test tube with 100% N2 and saturating for at least 50 min. The cells were perfused at a constant flow rate (2 ml/min). Meanwhile, the perfusing bath was covered by a relatively tight covering and bubbled with 100% N2 to prevent the oxygen in the air from diffusing into the perfuse solution. The oxygen tension in the bath could be reduced to about 2.66 kPa (20 mm Hg) in 3–5 min and was monitored with an ISO2 isolated dissolved oxygen meter (WPI, USA). 2.3. Protocol of experiments 2.3.1. Protocol of hypoxia and hypoxia + milrinone treatments Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to a hypoxia (100% N2, glucose-free) solution for 7 min (hypoxia). Next, isolated cells were perfused with a hypoxia (100% N2, glucose-free) solution containing 0.1 mM milrinone. 2.3.2. Protocol of hydrogen dioxide and hydrogen dioxide + milrinone treatments Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to Tyrode's solution with 0.3 mM hydrogen peroxide for 7 min (hydrogen peroxide). Next, Isolated cells were perfused with Tyrode's solution containing 0.3 mM hydrogen peroxide and 0.01 mM or 0.1 mM milrinone. 2.4. Electrical recordings Experiments were performed at room temperature (22–24 °C). Guinea pig ventricular myocytes were placed into a recording chamber that was bathed with Tyrode's solution, without or with drug(s), at a rate of 2 ml/min. The Tyrode solution contained (in mM): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, glucose 10, and HEPES 10 (pH 7.4). INaP was recorded in voltage clamp mode and action potential was
207
recorded in current clamp mode by using whole-cell patch-clamp techniques in guinea pig ventricular myocytes. Patch electrodes were pulled with a two-stage puller (PP-830, Narishige Group, Tokyo, Japan). For whole-cell recordings, their resistances were in the range of 1– 1.5 MΩ. To record the INaP, the intracellular pipette solution contained (in mM): CsCl2 120, CaCl2 1, MgCl2 5, Na2ATP 5, TEACl 10, EGTA 11, and HEPES 10 (pH 7.3). Capacitance and series resistances were adjusted to obtain minimal contribution of the capacitive transients. A 60% to 80% compensation of the series resistance was usually achieved without ringing. To record the action potential, the intracellular pipette solution contained (in mM): KCl 120, CaCl2 1, MgCl2 5, Na2ATP 5, EGTA 11, HEPES 10, and glucose 10 (pH 7.3). Currents were obtained with a Multiclamp 700B amplifier (Axon Instruments, Inc.USA), filtered at 2 kHz, digitized at 10 kHz, and stored on a computer hard disk for further analysis. 2.5. Drugs and reagents Collagenase type I and CsCl were obtained from Gibco (GIBCO TM, Invitrogen, Paisley, UK). Bovine serum albumin (BSA), HEPES and taurine were obtained from Roche (Basel, Switzerland). Tetrodotoxin (TTX) was purchased from Hebei Fisheries Research Institute (Qinhuangdao, China). Hydrogen peroxide was a production of Wuhan Zhongnan Chemical Reagent Co. (Wuhan, China). All other chemicals were purchased from Sigma Chemical (Saint Louis, Missouri, USA). Milrinone was used at 0.01 mM or 0.1 mM diluted from a stock of 100 mM in dimethylsulfoxide (DMSO). DMSO, when present, was at a concentration of 0.01% or 0.1%. Control experiments (n = 3) showed no effect of DMSO when present at 0.1%. 2.6. Data analysis Whole-cell recordings were analyzed using clampfit 9.0 (Axon Instruments, Inc.USA). Figures were plotted by Origin (V7.0, OriginLab Co., MA, USA). To eliminate the influence of INaT, all amplitudes of INaP were tested at 200 ms in depolarization testing pulse. Statistical significance between two groups and multiple groups were evaluated by Student's t-test and one-way analysis of variance (ANOVA), respectively. All values were expressed as mean ± S.D., and the number of cells (n) in each group was given. P b 0.05 was considered to be statistically significant. 3. Results 3.1. Confirmation of INa.P INa.P was recorded first in the absence and then in the presence of 2 μM TTX with 300 ms voltage steps from a holding potential of − 120 to − 20 mV. INa.T did not change obviously, while INa.P was almost blocked completely (n = 6), indicating that these currents were sodium currents (Fig. 1A). 3.2. Effects of milrinone on INa.P under normal condition Control value of INa.P was decreased by 0.01 mM milrinone or 0.1 mM milrinone (Table 1, Fig. 1B, C). Fig. 1D shows the magnitudes of INa. P at all voltages (before and after application of 0.1 mM milrinone) and 0.1 mM milrinone did not modify the voltage at which INa.P amplitude was maximal. 3.3. Effects of milrinone on increased INa.P induced by hypoxia Under hypoxic conditions, it was found that mean current density of INa.p increased, whereas milrinone at 0.1 mM reversed the increased INa.P (Table 1, Fig. 2A). Fig. 2D shows the I–V relationships of INa.P after the sequential handles of hypoxia and the additions of 0.1 mM
208
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
Fig. 1. Milrinone inhibited INa.P of guinea pig in ventricular myocytes. A, TTX (2 μM) abolished INa.P. B, Milrinone (0.01 mM) decreased INa.P. Myocytes were sequentially treated with no drug (control) and milrinone. C, Milrinone (0.1 mM) decreased INa.P. Myocytes were sequentially treated with no drug (control) and milrinone. D, Effect of milrinone (0.1 mM) bath application on current voltage relationship (n = 6). Values are expressed as mean ± S.D. * P b 0.05; † P b 0.01.
milrinone, without a shift of the voltage at which INa.P amplitude was maximal.
3.4. Effects of milrinone on increased INa.P induced by hydrogen dioxide With the presence of 0.3 mM hydrogen peroxide, we observed that mean current density of INa.p increased significantly, whereas 0.01 mM milrinon or 0.1 mM milrinone reversed the increased INa.P at different degrees (Table 1, Fig. 2B, C). Fig. 2E shows the I–V relationships of INa.P after the sequential handles of 0.3 mM hydrogen peroxide and the
Table 1 The effects of milrinone on INa.p in guinea pig ventricular myocytes. Treatment
INa.p, pA/pF
n
a. Control b. Milrinone (0.01 mM) a. Control b. Milrinone (0.1 mM) a. Control b. Hypoxia 7 min c. Milrinone (0.1 mM) a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.01 mM) a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.1 mM) a. Control b. cAMP (0.1 mM) a. Control + 1 μM H−89 b. Milrinone (0.1 mM)
− 0.325 ± 0.0340 − 0.265 ± 0.0314b − 0.330 ± 0.0535 − 0.244 ± 0.0292b c − 0.274 ± 0.0326 − 0.723 ± 0.0697b − 0.477 ± 0.0628b − 0.293 ± 0.0291 − 0.529 ± 0.0431b − 0.414 ± 0.0471b − 0.309 ± 0.0378 − 0.546 ± 0.0529b − 0.383 ± 0.0386b d − 0.330 ± 0.0354 − 0.219 ± 0.0399a − 0.350 ± 0.0319 − 0.337 ± 0.0244
7 7 6 6 6 6 6 7 7 7 6 6 6 6 6 6 6
Values are expressed as mean ± S.D. from n cells. aP b 0.05; bP b 0.01; cP b 0.05 versus control + 0.01 mM milrinone group; dP b 0.05 versus control + 0.3 mM hydrogen dioxide + 0.01 mM milrinone group.
additions of 0.1 mM milrinone, without a shift of the voltage at which INa.P amplitude was maximal. 3.5. Effects of cAMP on INa.P under normal condition Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to Tyrode's solution with 0.1 mM cAMP for 3 min (cAMP). The result showed that INa.p was significantly decreased by 0.1 mM cAMP (Table 1, Fig. 3A, C, D). 3.6. Effects of H-89 on milrinone In this group 1 μM H-89 (a highly selective PKA inhibitor) was added in pipette solution. Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to Tyrode's solution with 0.1 mM milrinone for 3 min (milrinone). We found that milrinone (0.1 mM) could not significantly decrease INa.P (Table 1, Fig. 3B, C, D). 3.7. Effect of milrinone on action potential waveforms Action potential was recorded in current clamp mode by using whole-cell patch-clamp techniques and was evoked by depolarizing current pulses (frequency 1 Hz, duration 5 ms, 1.5 times the threshold intensity) in guinea pig ventricular myocytes. Five groups were involved in this part. In group 1 and group 2, isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) for 10 min, then cells in group 1 were exposed to Tyrode's solution with 0.01 mM milrinone for 3 min and cells in group 2 were exposed to Tyrode's solution with 0.1 mM milrinone for 3 min. We observed that milrinone shortened APD90 and had no effect on the value of resting potential, action potential amplitude and Vmax (Table 2, Fig. 4A, B).
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
209
Fig. 2. Milrinone inhibited the increased INa.P induced by hypoxia or hydrogen dioxide. A, Milrinone (0.1 mM) attenuated INa.P induced by hypoxia. B, Milrinone (0.01 mM) attenuated INa.P induced by hydrogen dioxide. C, Milrinone (0.1 mM) attenuated INa.P induced by hydrogen dioxide. D, The I–V relationships of INa.P after the sequential handles of hypoxia and the additions of 0.1 mM milrinone. Values are expressed as mean ± S.D. *P b 0.05 versus control; † P b 0.01 versus control; # P b 0.01 versus hypoxia group. E, The I–V relationships of INa.P after the sequential additions of 0.3 mM hydrogen dioxide and 0.1 mM milrinone. Values are expressed as mean ± S.D. * P b 0.05 versus control; † P b 0.01 versus control; # P b 0.01 versus hydrogen dioxide group; § P b 0.05 versus hydrogen dioxide group.
In group 3, the myocytes were sequentially treated with no drug (control), hydrogen peroxide, and hydrogen peroxide plus 2 µM TTX. Hydrogen peroxide (0.3 mM) prolonged the APD90 at approximately
7 min, whereas TTX reversed the APD90 and had no effect on the value of resting potential, action potential amplitude and Vmax (Table 2, Fig. 4C).
Fig. 3. Inhibitory effects of milrinone on INa.P are mediated partly by cAMP pathway. A, cAMP (0.1 mM) decreased INa.P. B, H-89 (1 μM in pipette solution) antagonized the effects of milrinone on INa.P. C, Tendency of INa.P along time at a test potential of − 30 mV from a holding potential of − 120 mV is shown. D, Histograms show the mean current density of INaP under different conditions. Mean ± S.D., †P b 0.01 versus control group, *P b 0.01 versus 0.1 mM cAMP group, ** P b 0.01 versus 0.1 mM milrinone group.
210
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
Table 2 The effects of milrinone on action potential waveforms in guinea pig ventricular myocytes. Treatment
Resting potential (mV)
Action potential amplitude (mV)
Vmax(V/s)
APD90(ms)
a. Control b. Milrinone (0.01 mM)
− 80.9 ± 1.2 − 81.1 ± 1.2
130.6 ± 2.4 131.1 ± 2.1
236.6 ± 12.3 240.5 ± 13.7
265.0 ± 12.9 245.8 ± 13.2b
n=7 − 80.4 ± 1.0 − 80.7 ± 1.1
n=7 126.7 ± 2.2 128.7 ± 2.6
n=7 236.7 ± 22.9 239.2 ± 16.8
n=7 263.2 ± 13.9 238.4 ± 12.6b
n=7 − 80.8 ± 1.3 − 79.9 ± 0.8
n=7 122.6 ± 7.4 124.2 ± 5.0
n=7 231.3 ± 15.5 234.1 ± 14.5
n=7 258.4 ± 18.1 311.6 ± 20.4b
− 79.8 ± 0.8 n=7 − 81.7 ± 1.1 − 81.5 ± 1.2
127.6 ± 5.5 n=7 129.6 ± 5.7 130.2 ± 6.0
235.4 ± 10.5 n=7 234.9 ± 11.8 237.2 ± 12.2
233.1 ± 14.1b n=7 252.7 ± 11.7 324.0 ± 13.6b
− 81.9 ± 1.3
130.1 ± 6.3
230.7 ± 12.8
300.9 ± 14.6b
− 81.4 ± 1.3 n=7 − 81.4 ± 1.5 − 82.4 ± 1.4
129.5 ± 6.3 n=7 124.3 ± 6.6 125.3 ± 7.0
235.9 ± 10.7 n=7 243.9 ± 13.5 241.3 ± 11.0
236.9 ± 13.1b n=7 264.7 ± 14.3 336.6 ± 15.1b
− 81.9 ± 1.1
127.1 ± 5.8
245.9 ± 9.5
298.1 ± 15.2b
− 81.6 ± 1.0 n=7
125.3 ± 6.9 n=7
247.4 ± 11.5 n=7
245.2 ± 14.4b n=7
a. Control b. Milrinone (0.1 mM) a. Control b. Hydrogen dioxide (0.3 mM) c. 2 μM TTX a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.01 mM) d. 2 μM TTX a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.1 mM) d. 2 μM TTX
Vmax = maximum rate of depolarization; APD90 = action potential duration at 90% repolarization. Values are expressed as mean ± S.D. from n cells. aP b 0.05; bP b 0.01.
In group 4 and group 5, the myocytes were sequentially treated with no drug (control), hydrogen peroxide (7 min), hydrogen peroxide plus milrinone (0.01 mM in group 4 and 0.1 mM in group 5), and hydrogen
peroxide plus both milrinone (0.01 mM in group 4 and 0.1 mM in group 5) and 2 µM TTX. We observed that milrinone shortened the APD90 induced by hydrogen peroxide and had no effect on the value of resting potential, action potential amplitude and Vmax. Finally, TTX caused a further decurtation of APD90 and had no effect on the value of resting potential, action potential amplitude and Vmax (Table 2, Fig. 4D, E). 4. Discussion Ion regulation and the maintenance of ion gradients across the cell membrane are important for cell homeostasis. In cardiac tissue, ionic imbalance and current disorder can be precursors to the genesis of arrhythmias (Levi et al., 1997), which may degenerate to fibrillation and sudden cardiac death. Hypoxia-enhanced INa.P possibly causes ischemic arrhythmia in two ways. Firstly, as INa.P distributes mostly at the medium of ventricular myocardium (called M cells) and increases significantly during hypoxia, the increased INa.P prolongs the action potential plateau in M cells significantly while the plateau of endothelial and extima cells in ventricular myocardium changes slightly compared with that of M cells (Sakmann et al., 2000; Zygmunt et al., 2001). Therefore, the repolarization dispersion of the ventricular wall is enlarged and reentry arrhythmia is more likely to be induced (Antzelevitch, 2000). Increased INa.P and reductions in repolarizing K+ currents, whether caused by drugs or by heritable ion channel dysfunction, prolong the QT interval and act as major predisposing factors for torsade de pointes (TdP) in humans (Sasyniuk et al., 1989). Prolongation of the QT interval beyond a certain limit may herald proarrhythmic events (Shaffer et al., 2002; Zabel et al., 1998). Secondly, the increased INa.P raises the concentration of intracellular Na+ and consequently causes overload of intracellular Ca2+ through the reverse Na+–Ca2+ exchanger, finally inducing delayed afterdepolarization (DAD) or early afterdepolarization (EAD) (Song et al.,
Fig. 4. Milrinone shortened APD90 under normal conditions and the prolonged APD90 induced by hydrogen dioxide in guinea pig ventricular myocytes. A, The effects of 0.01 mM milrinone on action potential waveforms under normal condition. B, The effects of 0.1 mM milrinone on action potential waveforms under normal condition. C, The effects of 2 µM TTX on action potential waveforms induced by 0.3 mM hydrogen dioxide. D, The effects of 0.01 mM milrinone on action potential waveforms induced by 0.3 mM hydrogen dioxide, the myocytes being sequentially treated with no drug (control), hydrogen dioxide, hydrogen dioxide plus 0.01 mM milrinone, and hydrogen dioxide plus both 0.01 mM milrinone and 2 µM TTX. E, The effects of 0.1 mM milrinone on action potential waveforms induced by 0.3 mM hydrogen dioxide, the myocytes being sequentially treated with no drug (control), hydrogen dioxide, hydrogen dioxide plus 0.1 mM milrinone, and hydrogen dioxide plus both 0.1 mM milrinone and 2 µM TTX.
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
2008; Sugiyama and Hashimoto, 1999). Therefore, we hold that INa.P is a potential target for therapeutic intervention in ischaemia and heart failure (Belardinellia et al., 2004; Hale and Kloner, 2006; Song et al., 2006; Sossalla et al., 2008; Wang et al., 2008). Besides, milrinone increased the magnitude of the slow inward calcium current in voltage-clamped calf cardiac Purkinje fiber (Sutko et al., 1986) and it, when at 100 µM, enhanced the slowly activating component of delayed rectifier K+ current (IKs) in guinea pig sinoatrial (SA) node cells (Shimizu et al., 2002). The augmentation of IKs may contribute to the action potential duration shortening, but the augmentation of the slow inward calcium current is likely to bring about the action potential duration prolongation. In addition, milrinone had no effect on the hERG current up to 100 µM in hERG transfected CHO-K1 cells (Yunomae et al., 2007). In this study, milrinone effectively reduced INa.P under normal condition, hydrogen dioxide-induced INa.P and hypoxia-induced INa.P(Figs. 1, 2). This effect of milrinone is similar to that of TTX. Hydrogen dioxide, a species of reactive oxygen species, whose production is enhanced during ischemia-reperfusion of the heart, is produced as a by-product of oxidative metabolism, in which energy activation and electron reduction are involved (Kourie, 1998). The excessive amount of hydrogen dioxide induces INa.P in ventricular myocytes (Ma et al., 2005). Recent reports show that INa.P induces intracellular sodium overload, which promotes calcium overload via reverse NCX (Haigney et al., 1994; Song et al., 2006; Sossalla et al., 2008), resulting in cell injury (Marsh and Smith, 1991), ventricular electrical instability (Song et al., 2006), ventricular mechanical dysfunction (Belardinellia et al., 2004; Song et al., 2006; Wang et al., 2008) and serious arrhythmia (Thandroyen et al., 1991). In our experiment, hydrogen dioxide prolongs the action potential duration and 2 µM TTX almost eliminates the effect of hydrogen dioxide on the action potential duration (Fig. 4C). This indicates that the action potential duration prolonged by hydrogen dioxide probably results from an increased INa.P induced by hydrogen dioxide. Other studies have shown that increased INa.P not only prolongs action potential duration (Ward and Giles, 1997), but also causes EAD, which may explain, at least in part, the arrhythmogenic effect of hydrogen dioxide on cardiac myocytes (Song et al., 2006). In short, milrinone inhibits prolonged action potential duration associated with hydrogen dioxide-induced INa.P, which is consistent with previous study in guinea pig papillary muscles (Uemura et al., 1999). This shows that milrinone can attenuate exaggeration of transmural dispersion of repolarization and decrease ventricular electrical and mechanical dysfunction. Meanwhile, the inhibitive effect of milrinone on hypoxia or hydrogen dioxide-induced INa.P may reduce the rise in intracellular [Na+] and [Ca2+], which lowers the electrical and mechanical abnormalities associated with such conditions. Furthermore, results of other studies have shown that intracellular sodium overload and calcium overload caused by increased INa.P are more or less associated with pathogenic mechanism of ischemic cardiomyopathy (Saint, 2006), long-QT syndrome 3 (Crotti et al., 2008; Wu et al., 2004) and heart failure (Maltsev et al., 2007; Valdivia et al., 2005). Under these pathological conditions, digitalis poisoning (calcium overload) easily occurs. Thus, as a substitute, milrinone may be beneficial for patients associated with such conditions. Milrinone increases intracellular cAMP by inhibiting type III phosphodiesterase, with the result of a positive inotropic effect on the heart, vasodilatation in the periphery and reduction in ventricular relaxation time (Farah and Frangakis, 1989). Furthermore, in our study, we found while cAMP could inhibit INa.P, H-89 (a highly selective PKA inhibitor) can eliminate the inhibitive effect of milrinone on INa.P. This indicates that milrinone inhibits INa.P via activation of PKA. Another study reported that cAMP could cause a shift in the steady-state inactivation curve of sodium channnel to more negative potentials probably by altering the gating mechanism
211
of the Na+ channel via phosphorylation (caused by activation of PKA) of multiple sites on cardiac Na+ channels (Ono et al., 1989). We conclude that the inhibitive effect of milrinone on INa.P is associated with activation of cAMP-PKA signaling pathway. It has been well documented that milrinone did provide shortterm benefits for heart failure. And a study shows that INa.P is increased in heart failure ventricular cells from a canine pacing model of heart failure and also from explanted failing human hearts (Valdivia et al., 2005). But long-term studies with milrinone showed reduced survival, which is likely to be related to cardiac arrhythimias, possibly resulting from increased intracellular cAMP (Cruickshank, 1993). Despite these data, some physicians have proposed that the regularly scheduled intermittent use of intravenous positive inotropic drugs (e.g., dobutamine or milrinone) in a supervised outpatient setting might be associated with some clinical benefits (Cesario et al., 1998; Marius-Nunez et al., 1996). In summary, milrinone inhibits INa.P under normal condition, hydrogen dioxide- induced INa.P, hypoxia-induced INa.P and the prolonged action potential duration associated with hydrogen dioxide- induced INa.P in ventricular myocytes, which results in attenuated exaggeration of transmural dispersion of repolarization, the electrical instability (formation of EADs and arrhythmia) and mechanical dysfunction. The inhibitive effect of milrinone on hypoxia or hydrogen dioxide-induced INa.P may reduce the rise in intracellular [Na+] and [Ca2+], which lowers the electrical and mechanical abnormalities associated with these conditions. All these effects are associated with the activation of cAMP-PKA signaling pathway. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30670764). References Antzelevitch, C., 2000. Electrical heterogeneity, cardiac arrhythmias, and the sodium channel. Circ. Res. 87, 964–965. Ashamalla, S.M., Navarro, D., Ward, C.A., 2001. Gradient of sodium current across the left ventricular wall of adult rat hearts. J. Physiol. 536, 439–443. Belardinellia, L., Antzelevitch, C., Fraserc, H., 2004. Inhibition of late (sustained/ persistent) sodium current: a potential drug target to reduce intracellular sodiumdependent calcium overload and its detrimental effects on cardiomyocyte function. Eur. Heart J. 6, 13–17. Cesario, D., Clark, J., Maisel, A., 1998. Beneficial effects of intermittent home administration of the inotrope/vasodilator milrinone in patients with end-stage congestive heart failure: a preliminary study. Am. Heart J. 135, 121–129. Chen, E.P., Bittner, H.B., Davis Jr., R.D., Van Trigt III, P., 1997. Milrinone improves pulmonary hemodynamics and right ventricular function in chronic pulmonary hypertension. Ann. Thorac. Surg. 63, 814–821. Crotti, L., Celano, G., Dagradi, F., Schwartz, P.J., 2008. Congenital long QT syndrome. Orphanet. J. Rare Dis. 3, 18. Cruickshank, 1993. Phosphodiesterase III inhibitors: long-term risks and short-term benefits. Cardiovasc. Drugs Ther. 7, 655–660. Farah, A.E., Frangakis, C.J., 1989. Studies on the mechanism of action of the bipyridine milrinone on the heart. Basic Res. Cardiol. 84, 85–103. Friedman, J.E., Haddad, G.G., 1994. Anoxia induces an increase in intracellular sodium in rat central neurons in vitro. Brain Res. 663, 329–334. Ju, Y.K., Saint, D.A., Gage, P.W., 1996. Hypoxia increases persistent sodium current in rat ventricular myocytes. J. Physiol. 497, 337–347. Haigney, M.C., Lakatta, E.G., Stern, M.D., Silverman, H.S., 1994. Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading. Circulation 90, 391–399. Hale, S.L., Kloner, R.A., 2006. Ranolazine, an inhibitor of the late sodium channel current, reduces postischemic myocardial dysfunction in the rabbit. J. Cardiovasc. Pharmacol. Ther. 11, 249–255. Hoffman, T.M., Wernovsky, G., Atz, A.M., Bailey, J.M., Akbary, A., Kocsis, J.F., Nelson, D.P., Chang, A.C., Kulik, T.J., Spray, T.L., Wessel, D.L., 2002. Prophylactic intravenous use of milrinone after cardiac operation in pediatrics (PRIMACORP) study. Am. Heart J. 143, 15–21. Hoffman, T.M., Wernovsky, G., Atz, A.M., Kulik, T.J., Nelson, D.P., Chang, A.C., Bailey, J.M., Akbary, A., Kocsis, J.F., Kaczmarek, R., Spray, T.L., Wessel, D.L., 2003. Efficacy and safety of milrinone in preventing low cardiac output syndrome in infants and children after corrective surgery for congenital heart disease. Circulation 107, 996–1002. Honen, B.N., Saint, D.A., 2002. Heterogeneity of the properties of INa in epicardial and endocardial cells of rat ventricle. Clin. Exp. Pharmacol. Physiol. 29, 161–166.
212
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
Kiyosue, T., Arita, M., 1989. Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes. Circ. Res. 64, 389–397. Kourie, J.I., 1998. Interaction of reactive oxygen species with ion transport mechanisms. Am. J. Physiol. 275, 1–24. Levi, A.J., Dalton, G.R., Hancox, J.C., Mitcheson, J.S., Issberner, J., Bates, J.A., Evans, S.J., Howarth, F.C., Hobai, I.A., Jones, J.V., 1997. Role of intracellular sodium overload in the genesis of cardiac arrhythmias. J. Cardiovasc. Electrophysiol. 8, 700–721. Ma, J.H., Luo, A.T., Zhang, P.H., 2005. Effect of hydrogen peroxide on persistent sodium current in guinea pig ventricular myocytes. Acta Pharmacol. Sin. 26, 828–834. Maltsev, V.A., Silverman, N., Sabbah, H.N., Undrovinas, A.I., 2007. Chronic heart failure slows late sodium current in human and canine ventricular myocytes: implications for repolarization variability. Eur. J. Heart Fail. 9, 219–227. Marius-Nunez, A.L., Heaney, L., Fernandez, R.N., Clark, W.A., Ranganini, A., Silber, E., Denes, P., 1996. Intermittent inotropic therapy in an outpatient setting: a costeffective therapeutic modality in patients with refractory heart failure. Am. Heart J. 132, 805–808. Marsh, J.D., Smith, T.S., 1991. Calcium overload and ischemic myocardial injury. Circulation 83, 709–711. Niemann, J.T., Garner, D., Khaleeli, E., Lewis, R.J., 2003. Milrinone facilitates resuscitation from cardiac arrest and attenuates postresuscitation myocardial dysfunction. Circulation 108, 3031–3035. Ono, K., Kiyosue, T., Arita, M., 1989. Isoproterenol, DBcAMP, and forskolin inhibit cardiac sodium current. Am. J. Physiol. 256, 1131–1137. Saint, D.A., 2006. The role of the persistent Na (+) current during cardiac ischemia and hypoxia. J. Cardiovasc. Electrophysiol. 17, S96–S103. Sakmann, B.F., Spindler, A.J., Bryant, S.M., Linz, K.W., Noble, D., 2000. Distribution of a persistent sodium current across the ventricular wall in guinea pigs. Circ. Res. 87, 910–914. Sasyniuk, B.I., Valois, M., Toy, W., 1989. Recent advances in understanding the mechanisms of drug-induced torsades de pointes arrhythmias. Am. J. Cardiol. 64, 29J–32J. Shaffer, D., Singer, S., Korvick, J., Honig, P., 2002. Concomitant risk factors in reports of torsades de pointes associated with macrolide use: review of the United States Food and Drug Administration Adverse Event Reporting System. Clin. Infect. Dis. 35, 197–200. Shimizu, K., Shintani, Y., Ding, W.G., Matsuura, H., Bamba, T., 2002. Potentiation of slow component of delayed rectifier K(+) current by cGMP via two distinct mechanisms: inhibition of phosphodiesterase 3 and activation of protein kinase G. Br. J. Pharmacol. 137, 127–137. Shipley, J.B., Tolman, D., Hastillo, A., Hess, M.L., 1996. Milrinone: basic and clinical pharmacology and acute and chronic management. Am. J. Med. Sci. 311, 286–291. Simonton, C.A., Chatterjee, K., Cody, R.J., Kubo, S.H., Leonard, D., Daly, P., Rutman, H., 1985. Milrinone in congestive heart failure: acute and chronic hemodynamic and clinical evaluation. J. Am. Coll. Cardiol. 6, 453–459.
Song, Y., Shryock, J.C., Luiz Belardinelli, L., 2008. An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes. Am. J. Physiol. Heart Circ. Physiol. 294, H2031–H2039. Song, Y., Shryock, J.C., Wagner, S., Maier, L.S., Belardinelli, L., 2006. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J. Pharmacol. Exp. Ther. 318, 214–222. Sossalla, S., Wagner, S., Rasenack, E.C., Ruff, H., Weber, S.L., Schöndube, F.A., Tirilomis, T., Tenderich, G., Hasenfuss, G., Belardinelli, L., Maier, L.S., 2008. Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts — role of late sodium current and intracellular ion accumulation. J. Mol. Cell. Cardiol. 45, 32–43. Sugiyama, A., Hashimoto, K., 1999. Can the MAP technique be applied to detect delayed afterdepolarization? Electrophysiologic and pharmacologic evidence. J. Cardiovasc. Pharmacol. 34, 46–52. Sutko, J.L., Kenyon, J.L., Reeves, J.P., 1986. Effects of amrinone and milrinone on calcium influx into the myocardium. Circulation 73, III52–III58. Thandroyen, F.T., Morris, A.C., Hagler, H.K., Ziman, B., Pai, L., Willerson, J.T., Buja, L.M., 1991. Intracellular calcium transients and arrhythmia in isolated heart cells. Circ. Res. 69, 810–819. Uemura, H., Sakamoto, N., Nakaya, H., 1999. Electropharmacological effects of UK-1745, a novel cardiotonic drug, in guinea-pig ventricular myocytes. Eur. J. Pharmacol. 383, 361–371. Valdivia, C.R., Chu, W.W., Pu, J., Foell, J.D., Haworth, R.A., Wolff, M.R., Kamp, T.J., Makielski, J.C., 2005. Increased late sodium current in myocytes from a canine heart failure model and from failing human heart. J. Mol. Cell. Cardiol. 38, 475–483. Wang, L., Lopaschuk, G.D., Clanachan, A.S., 2008. H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation. J. Mol. Cell. Cardiol. 45, 787–795. Ward, C.A., Giles, W.R., 1997. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J. Physiol. 500, 631–642. Wu, L., Shryock, J.C., Song, Y., Li, Y., Antzelevitch, C., Belardinelli, L., 2004. Antiarrhythmic effects of ranolazine in a guinea pig in vitro model of long-QT syndrome. J. Pharmacol. Exp. Ther. 310, 599–605. Yunomae, K., Ichisaki, S., Matsuo, J., Nagayama, S., Fukuzaki, K., Nagata, R., Kito, G., 2007. Effects of phosphodiesterase (PDE) inhibitors on human ether-a-go-go related gene (hERG) channel activity. J. Appl. Toxicol. 27, 78–85. Zabel, M., Lichtlen, P.R., Haverich, A., Franz, M.R., 1998. Comparison of ECG variables of dispersion of ventricular repolarization with direct myocardial repolarization measurements in the human heart. J. Cardiovasc. Electrophysiol. 9, 1279–1284. Zygmunt, A.C., Eddlestone, G.T., Thomas, G.P., Nesterenko, V.V., Antzelevitch, C., 2001. Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle. Am. J. Physiol., Heart Circ. Physiol. 281, 689–697.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Milrinone inhibits hypoxia or hydrogen dioxide-induced persistent sodium current in ventricular myocytes Jie Zheng, Jihua Ma ⁎, Peihua Zhang, Liangkun Hu, Xinrong Fan, Qiong Tang Cardio-Electrophysiological Research Laboratory, Medical College, Wuhan University of Science and Technology, Wuhan, Hubei, 430081, China
a r t i c l e
i n f o
Article history: Received 17 January 2009 Received in revised form 28 May 2009 Accepted 9 June 2009 Available online 21 June 2009 Keywords: Milrinone Persistent sodium current Hypoxia Hydrogen peroxide Patch-clamp
a b s t r a c t Much evidence indicates that increased persistent sodium current (INa.P) is associated with cellular calcium overload and INa.P is considered to be a potential target for therapeutic intervention in ischaemia and heart failure. By inhibiting type III phosphodiesterase, milrinone increases intracellular cyclic adenosine monophosphate (cAMP), with a positive inotropic effect. However, the effect of milrinone on increased INa.P under pathological conditions remains unknown. Accordingly, we investigated the effect of milrinone on increased INa.P induced by hypoxia or hydrogen dioxide in guinea pig ventricular myocytes. While milrinone (0.01 mM or 0.1 mM) or cAMP (0.1 mM) decreased INa.P respectively in control condition, application of 1 μM H-89, a selective cAMP-dependant protein kinase inhibitor, prevented the effect of 0.1 mM milrinone in control condition. Milrinone (0.1 mM) reduced the increased INa.P induced by hypoxia. Furthermore, 0.01 mM or 0.1 mM milrinone reduced the enhanced INa.P induced by 0.3 mM hydrogen peroxide. In addition, 0.01 mM or 0.1 mM milrinone shortened action potential duration at 90% repolarization (APD90). Bath application of 0.3 mM hydrogen dioxide markedly prolonged APD90, while 2 μM tetrodotoxin (TTX) reversed the prolonged APD90. In the other two groups, 0.01 mM or 0.1 mM milrinone shortened the prolonged APD90 induced by 0.3 mM hydrogen peroxide, ultimately 2 μM TTX causing a further decurtation of APD90. These findings demonstrate that milrinone inhibited INa.P under normal condition, hypoxia or hydrogen dioxide-induced INa. P, and the APD90 prolonged by hydrogen dioxide-induced INa.P in ventricular myocytes, which is associated with the mechanism of milrinone increasing intracellular cAMP. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sodium current is an important ion current in cardiac tissues. Under normal condition the amplitude of INaP is much smaller than that of INaT, which is responsible for the upstroke and conduction of the action potential. However, INaP plays an important role in maintaining the plateau of action potential, determining action potential duration (Kiyosue and Arita, 1989), altering [Na+]i (Saint, 2006), transmurally dispersing repolarization and developing cardiac arrhythmias (Ashamalla et al., 2001; Honen and Saint, 2002; Sakmann et al., 2000; Zygmunt et al., 2001). Since Ju et al. (1996) reported that hypoxia could increase persistent sodium current in rat ventricular myocytes, researchers have been paying increasingly attention to INaP. Several studies indicated that a rise of [Na+]i is caused by the increased INaP induced by hypoxia or reactive oxygen species and intracellular calcium overload caused by reactive oxygen species, which results in cell injury or death, results from a rise in [Na+]i followed by Ca2+ influx via the reverse mode of the Na+–Ca2+ exchanger (NCX) (Friedman and Haddad, 1994; Haigney et al., 1994; Song et al., 2006; Wang et al.,
⁎ Corresponding author. Tel./fax: +86 027 68862109. E-mail address: [email protected] (J. Ma). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.06.021
2008). Furthermore, increased INaP causes a prolongation of action potential duration, leading to arrhythmic activity and contractile dysfunction (Maltsev et al., 2007; Song et al., 2006; Wang et al., 2008). Therefore, inhibition of INaP is considered to be a potential target for therapeutic intervention in ischaemia and heart failure (Belardinellia et al., 2004; Hale and Kloner, 2006; Song et al., 2006; Sossalla et al., 2008; Wang et al., 2008). Milrinone represents a second generation of phosphodiesterase inhibitor. By inhibiting Type III phosphodiesterase, milrinone increases intracellular cyclic adenosine monophosphate (cAMP), resulting in a positive inotropic effect on the heart, vasodilatation in the periphery and reduction in ventricular relaxation time (Farah and Frangakis, 1989), without worsening major determinants of myocardial oxygen demand (Niemann et al., 2003). Milrinone is approved for intravenous administration in the treatment of congestive heart failure (Simonton et al., 1985), decompensated congestive heart failure (Shipley et al., 1996) and pulmonary hypertension (Chen et al., 1997). Furthermore, it is used for preventing low cardiac output syndrome after cardiac surgery (Hoffman et al., 2002, 2003). The positive effect of milrinone on cardiac mechanical activity has been reported widely. However, the effect of milrinone on cardiac electrical activity has been seldom studied and its effect on INaP has not been reported at all. While it is widely accepted that milrinone increases
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
intracellular cAMP, our study revealed cAMP can distinctly inhibit INaP. It is significant to investigate the effect of milrinone on INaP for further interpreting pharmacological mechanism of milrinone and expanding the scope of application of milrinone. 2. Materials and methods 2.1. Isolation of ventricular myocytes Adult guinea pigs (250–300 g, of either sex, grade II, Experiment Animal Center of Wuhan University of Science and Technology, Wuhan, China) were anesthetized with pentobarbital sodium (30 mg/kg, i.p.) 20 min after an intraperitoneal injection of 2000 U of heparin. Hearts were excised rapidly and perfused retrogradely on a Langendorff apparatus with a Ca2+-free Tyrode's solution for 5 min before the perfusate was switched to an enzyme-containing solution [0.1 g/l collagenase type I, 0.01 g/l protease E, 0.5 g/l bovine serum albumin (BSA) in the same solution] for 5 min. The perfusate was finally changed to KB solution containing (mM): KOH 70, taurine 20, glutamic acid 50, KCl 40, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, and glucose 10, pH 7.4, for 5 min. These perfusates were bubbled with 100% O2 and maintained at 37 °C. The ventricles were cut into small chunks and gently agitated in KB solution. The cells were filtered through nylon mesh and stored in KB solution at 4 °C. All procedures met the guide for the care and use of laboratory animals regulated by Administrative Regulation of Laboratory Animals of Hubei Province. 2.2. Induction of hypoxia The cell being examined was continuously perfused with extracellular solution flowing through a small plastic tube from a test tube. Hypoxia was achieved by bubbling the perfuse solution (normal Tyrode's solution without glucose) in this test tube with 100% N2 and saturating for at least 50 min. The cells were perfused at a constant flow rate (2 ml/min). Meanwhile, the perfusing bath was covered by a relatively tight covering and bubbled with 100% N2 to prevent the oxygen in the air from diffusing into the perfuse solution. The oxygen tension in the bath could be reduced to about 2.66 kPa (20 mm Hg) in 3–5 min and was monitored with an ISO2 isolated dissolved oxygen meter (WPI, USA). 2.3. Protocol of experiments 2.3.1. Protocol of hypoxia and hypoxia + milrinone treatments Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to a hypoxia (100% N2, glucose-free) solution for 7 min (hypoxia). Next, isolated cells were perfused with a hypoxia (100% N2, glucose-free) solution containing 0.1 mM milrinone. 2.3.2. Protocol of hydrogen dioxide and hydrogen dioxide + milrinone treatments Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to Tyrode's solution with 0.3 mM hydrogen peroxide for 7 min (hydrogen peroxide). Next, Isolated cells were perfused with Tyrode's solution containing 0.3 mM hydrogen peroxide and 0.01 mM or 0.1 mM milrinone. 2.4. Electrical recordings Experiments were performed at room temperature (22–24 °C). Guinea pig ventricular myocytes were placed into a recording chamber that was bathed with Tyrode's solution, without or with drug(s), at a rate of 2 ml/min. The Tyrode solution contained (in mM): NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, glucose 10, and HEPES 10 (pH 7.4). INaP was recorded in voltage clamp mode and action potential was
207
recorded in current clamp mode by using whole-cell patch-clamp techniques in guinea pig ventricular myocytes. Patch electrodes were pulled with a two-stage puller (PP-830, Narishige Group, Tokyo, Japan). For whole-cell recordings, their resistances were in the range of 1– 1.5 MΩ. To record the INaP, the intracellular pipette solution contained (in mM): CsCl2 120, CaCl2 1, MgCl2 5, Na2ATP 5, TEACl 10, EGTA 11, and HEPES 10 (pH 7.3). Capacitance and series resistances were adjusted to obtain minimal contribution of the capacitive transients. A 60% to 80% compensation of the series resistance was usually achieved without ringing. To record the action potential, the intracellular pipette solution contained (in mM): KCl 120, CaCl2 1, MgCl2 5, Na2ATP 5, EGTA 11, HEPES 10, and glucose 10 (pH 7.3). Currents were obtained with a Multiclamp 700B amplifier (Axon Instruments, Inc.USA), filtered at 2 kHz, digitized at 10 kHz, and stored on a computer hard disk for further analysis. 2.5. Drugs and reagents Collagenase type I and CsCl were obtained from Gibco (GIBCO TM, Invitrogen, Paisley, UK). Bovine serum albumin (BSA), HEPES and taurine were obtained from Roche (Basel, Switzerland). Tetrodotoxin (TTX) was purchased from Hebei Fisheries Research Institute (Qinhuangdao, China). Hydrogen peroxide was a production of Wuhan Zhongnan Chemical Reagent Co. (Wuhan, China). All other chemicals were purchased from Sigma Chemical (Saint Louis, Missouri, USA). Milrinone was used at 0.01 mM or 0.1 mM diluted from a stock of 100 mM in dimethylsulfoxide (DMSO). DMSO, when present, was at a concentration of 0.01% or 0.1%. Control experiments (n = 3) showed no effect of DMSO when present at 0.1%. 2.6. Data analysis Whole-cell recordings were analyzed using clampfit 9.0 (Axon Instruments, Inc.USA). Figures were plotted by Origin (V7.0, OriginLab Co., MA, USA). To eliminate the influence of INaT, all amplitudes of INaP were tested at 200 ms in depolarization testing pulse. Statistical significance between two groups and multiple groups were evaluated by Student's t-test and one-way analysis of variance (ANOVA), respectively. All values were expressed as mean ± S.D., and the number of cells (n) in each group was given. P b 0.05 was considered to be statistically significant. 3. Results 3.1. Confirmation of INa.P INa.P was recorded first in the absence and then in the presence of 2 μM TTX with 300 ms voltage steps from a holding potential of − 120 to − 20 mV. INa.T did not change obviously, while INa.P was almost blocked completely (n = 6), indicating that these currents were sodium currents (Fig. 1A). 3.2. Effects of milrinone on INa.P under normal condition Control value of INa.P was decreased by 0.01 mM milrinone or 0.1 mM milrinone (Table 1, Fig. 1B, C). Fig. 1D shows the magnitudes of INa. P at all voltages (before and after application of 0.1 mM milrinone) and 0.1 mM milrinone did not modify the voltage at which INa.P amplitude was maximal. 3.3. Effects of milrinone on increased INa.P induced by hypoxia Under hypoxic conditions, it was found that mean current density of INa.p increased, whereas milrinone at 0.1 mM reversed the increased INa.P (Table 1, Fig. 2A). Fig. 2D shows the I–V relationships of INa.P after the sequential handles of hypoxia and the additions of 0.1 mM
208
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
Fig. 1. Milrinone inhibited INa.P of guinea pig in ventricular myocytes. A, TTX (2 μM) abolished INa.P. B, Milrinone (0.01 mM) decreased INa.P. Myocytes were sequentially treated with no drug (control) and milrinone. C, Milrinone (0.1 mM) decreased INa.P. Myocytes were sequentially treated with no drug (control) and milrinone. D, Effect of milrinone (0.1 mM) bath application on current voltage relationship (n = 6). Values are expressed as mean ± S.D. * P b 0.05; † P b 0.01.
milrinone, without a shift of the voltage at which INa.P amplitude was maximal.
3.4. Effects of milrinone on increased INa.P induced by hydrogen dioxide With the presence of 0.3 mM hydrogen peroxide, we observed that mean current density of INa.p increased significantly, whereas 0.01 mM milrinon or 0.1 mM milrinone reversed the increased INa.P at different degrees (Table 1, Fig. 2B, C). Fig. 2E shows the I–V relationships of INa.P after the sequential handles of 0.3 mM hydrogen peroxide and the
Table 1 The effects of milrinone on INa.p in guinea pig ventricular myocytes. Treatment
INa.p, pA/pF
n
a. Control b. Milrinone (0.01 mM) a. Control b. Milrinone (0.1 mM) a. Control b. Hypoxia 7 min c. Milrinone (0.1 mM) a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.01 mM) a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.1 mM) a. Control b. cAMP (0.1 mM) a. Control + 1 μM H−89 b. Milrinone (0.1 mM)
− 0.325 ± 0.0340 − 0.265 ± 0.0314b − 0.330 ± 0.0535 − 0.244 ± 0.0292b c − 0.274 ± 0.0326 − 0.723 ± 0.0697b − 0.477 ± 0.0628b − 0.293 ± 0.0291 − 0.529 ± 0.0431b − 0.414 ± 0.0471b − 0.309 ± 0.0378 − 0.546 ± 0.0529b − 0.383 ± 0.0386b d − 0.330 ± 0.0354 − 0.219 ± 0.0399a − 0.350 ± 0.0319 − 0.337 ± 0.0244
7 7 6 6 6 6 6 7 7 7 6 6 6 6 6 6 6
Values are expressed as mean ± S.D. from n cells. aP b 0.05; bP b 0.01; cP b 0.05 versus control + 0.01 mM milrinone group; dP b 0.05 versus control + 0.3 mM hydrogen dioxide + 0.01 mM milrinone group.
additions of 0.1 mM milrinone, without a shift of the voltage at which INa.P amplitude was maximal. 3.5. Effects of cAMP on INa.P under normal condition Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to Tyrode's solution with 0.1 mM cAMP for 3 min (cAMP). The result showed that INa.p was significantly decreased by 0.1 mM cAMP (Table 1, Fig. 3A, C, D). 3.6. Effects of H-89 on milrinone In this group 1 μM H-89 (a highly selective PKA inhibitor) was added in pipette solution. Isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) and were then exposed to Tyrode's solution with 0.1 mM milrinone for 3 min (milrinone). We found that milrinone (0.1 mM) could not significantly decrease INa.P (Table 1, Fig. 3B, C, D). 3.7. Effect of milrinone on action potential waveforms Action potential was recorded in current clamp mode by using whole-cell patch-clamp techniques and was evoked by depolarizing current pulses (frequency 1 Hz, duration 5 ms, 1.5 times the threshold intensity) in guinea pig ventricular myocytes. Five groups were involved in this part. In group 1 and group 2, isolated cells were perfused with Tyrode's solution saturated with 100% O2 (control) for 10 min, then cells in group 1 were exposed to Tyrode's solution with 0.01 mM milrinone for 3 min and cells in group 2 were exposed to Tyrode's solution with 0.1 mM milrinone for 3 min. We observed that milrinone shortened APD90 and had no effect on the value of resting potential, action potential amplitude and Vmax (Table 2, Fig. 4A, B).
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
209
Fig. 2. Milrinone inhibited the increased INa.P induced by hypoxia or hydrogen dioxide. A, Milrinone (0.1 mM) attenuated INa.P induced by hypoxia. B, Milrinone (0.01 mM) attenuated INa.P induced by hydrogen dioxide. C, Milrinone (0.1 mM) attenuated INa.P induced by hydrogen dioxide. D, The I–V relationships of INa.P after the sequential handles of hypoxia and the additions of 0.1 mM milrinone. Values are expressed as mean ± S.D. *P b 0.05 versus control; † P b 0.01 versus control; # P b 0.01 versus hypoxia group. E, The I–V relationships of INa.P after the sequential additions of 0.3 mM hydrogen dioxide and 0.1 mM milrinone. Values are expressed as mean ± S.D. * P b 0.05 versus control; † P b 0.01 versus control; # P b 0.01 versus hydrogen dioxide group; § P b 0.05 versus hydrogen dioxide group.
In group 3, the myocytes were sequentially treated with no drug (control), hydrogen peroxide, and hydrogen peroxide plus 2 µM TTX. Hydrogen peroxide (0.3 mM) prolonged the APD90 at approximately
7 min, whereas TTX reversed the APD90 and had no effect on the value of resting potential, action potential amplitude and Vmax (Table 2, Fig. 4C).
Fig. 3. Inhibitory effects of milrinone on INa.P are mediated partly by cAMP pathway. A, cAMP (0.1 mM) decreased INa.P. B, H-89 (1 μM in pipette solution) antagonized the effects of milrinone on INa.P. C, Tendency of INa.P along time at a test potential of − 30 mV from a holding potential of − 120 mV is shown. D, Histograms show the mean current density of INaP under different conditions. Mean ± S.D., †P b 0.01 versus control group, *P b 0.01 versus 0.1 mM cAMP group, ** P b 0.01 versus 0.1 mM milrinone group.
210
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
Table 2 The effects of milrinone on action potential waveforms in guinea pig ventricular myocytes. Treatment
Resting potential (mV)
Action potential amplitude (mV)
Vmax(V/s)
APD90(ms)
a. Control b. Milrinone (0.01 mM)
− 80.9 ± 1.2 − 81.1 ± 1.2
130.6 ± 2.4 131.1 ± 2.1
236.6 ± 12.3 240.5 ± 13.7
265.0 ± 12.9 245.8 ± 13.2b
n=7 − 80.4 ± 1.0 − 80.7 ± 1.1
n=7 126.7 ± 2.2 128.7 ± 2.6
n=7 236.7 ± 22.9 239.2 ± 16.8
n=7 263.2 ± 13.9 238.4 ± 12.6b
n=7 − 80.8 ± 1.3 − 79.9 ± 0.8
n=7 122.6 ± 7.4 124.2 ± 5.0
n=7 231.3 ± 15.5 234.1 ± 14.5
n=7 258.4 ± 18.1 311.6 ± 20.4b
− 79.8 ± 0.8 n=7 − 81.7 ± 1.1 − 81.5 ± 1.2
127.6 ± 5.5 n=7 129.6 ± 5.7 130.2 ± 6.0
235.4 ± 10.5 n=7 234.9 ± 11.8 237.2 ± 12.2
233.1 ± 14.1b n=7 252.7 ± 11.7 324.0 ± 13.6b
− 81.9 ± 1.3
130.1 ± 6.3
230.7 ± 12.8
300.9 ± 14.6b
− 81.4 ± 1.3 n=7 − 81.4 ± 1.5 − 82.4 ± 1.4
129.5 ± 6.3 n=7 124.3 ± 6.6 125.3 ± 7.0
235.9 ± 10.7 n=7 243.9 ± 13.5 241.3 ± 11.0
236.9 ± 13.1b n=7 264.7 ± 14.3 336.6 ± 15.1b
− 81.9 ± 1.1
127.1 ± 5.8
245.9 ± 9.5
298.1 ± 15.2b
− 81.6 ± 1.0 n=7
125.3 ± 6.9 n=7
247.4 ± 11.5 n=7
245.2 ± 14.4b n=7
a. Control b. Milrinone (0.1 mM) a. Control b. Hydrogen dioxide (0.3 mM) c. 2 μM TTX a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.01 mM) d. 2 μM TTX a. Control b. Hydrogen dioxide (0.3 mM) c. Milrinone (0.1 mM) d. 2 μM TTX
Vmax = maximum rate of depolarization; APD90 = action potential duration at 90% repolarization. Values are expressed as mean ± S.D. from n cells. aP b 0.05; bP b 0.01.
In group 4 and group 5, the myocytes were sequentially treated with no drug (control), hydrogen peroxide (7 min), hydrogen peroxide plus milrinone (0.01 mM in group 4 and 0.1 mM in group 5), and hydrogen
peroxide plus both milrinone (0.01 mM in group 4 and 0.1 mM in group 5) and 2 µM TTX. We observed that milrinone shortened the APD90 induced by hydrogen peroxide and had no effect on the value of resting potential, action potential amplitude and Vmax. Finally, TTX caused a further decurtation of APD90 and had no effect on the value of resting potential, action potential amplitude and Vmax (Table 2, Fig. 4D, E). 4. Discussion Ion regulation and the maintenance of ion gradients across the cell membrane are important for cell homeostasis. In cardiac tissue, ionic imbalance and current disorder can be precursors to the genesis of arrhythmias (Levi et al., 1997), which may degenerate to fibrillation and sudden cardiac death. Hypoxia-enhanced INa.P possibly causes ischemic arrhythmia in two ways. Firstly, as INa.P distributes mostly at the medium of ventricular myocardium (called M cells) and increases significantly during hypoxia, the increased INa.P prolongs the action potential plateau in M cells significantly while the plateau of endothelial and extima cells in ventricular myocardium changes slightly compared with that of M cells (Sakmann et al., 2000; Zygmunt et al., 2001). Therefore, the repolarization dispersion of the ventricular wall is enlarged and reentry arrhythmia is more likely to be induced (Antzelevitch, 2000). Increased INa.P and reductions in repolarizing K+ currents, whether caused by drugs or by heritable ion channel dysfunction, prolong the QT interval and act as major predisposing factors for torsade de pointes (TdP) in humans (Sasyniuk et al., 1989). Prolongation of the QT interval beyond a certain limit may herald proarrhythmic events (Shaffer et al., 2002; Zabel et al., 1998). Secondly, the increased INa.P raises the concentration of intracellular Na+ and consequently causes overload of intracellular Ca2+ through the reverse Na+–Ca2+ exchanger, finally inducing delayed afterdepolarization (DAD) or early afterdepolarization (EAD) (Song et al.,
Fig. 4. Milrinone shortened APD90 under normal conditions and the prolonged APD90 induced by hydrogen dioxide in guinea pig ventricular myocytes. A, The effects of 0.01 mM milrinone on action potential waveforms under normal condition. B, The effects of 0.1 mM milrinone on action potential waveforms under normal condition. C, The effects of 2 µM TTX on action potential waveforms induced by 0.3 mM hydrogen dioxide. D, The effects of 0.01 mM milrinone on action potential waveforms induced by 0.3 mM hydrogen dioxide, the myocytes being sequentially treated with no drug (control), hydrogen dioxide, hydrogen dioxide plus 0.01 mM milrinone, and hydrogen dioxide plus both 0.01 mM milrinone and 2 µM TTX. E, The effects of 0.1 mM milrinone on action potential waveforms induced by 0.3 mM hydrogen dioxide, the myocytes being sequentially treated with no drug (control), hydrogen dioxide, hydrogen dioxide plus 0.1 mM milrinone, and hydrogen dioxide plus both 0.1 mM milrinone and 2 µM TTX.
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
2008; Sugiyama and Hashimoto, 1999). Therefore, we hold that INa.P is a potential target for therapeutic intervention in ischaemia and heart failure (Belardinellia et al., 2004; Hale and Kloner, 2006; Song et al., 2006; Sossalla et al., 2008; Wang et al., 2008). Besides, milrinone increased the magnitude of the slow inward calcium current in voltage-clamped calf cardiac Purkinje fiber (Sutko et al., 1986) and it, when at 100 µM, enhanced the slowly activating component of delayed rectifier K+ current (IKs) in guinea pig sinoatrial (SA) node cells (Shimizu et al., 2002). The augmentation of IKs may contribute to the action potential duration shortening, but the augmentation of the slow inward calcium current is likely to bring about the action potential duration prolongation. In addition, milrinone had no effect on the hERG current up to 100 µM in hERG transfected CHO-K1 cells (Yunomae et al., 2007). In this study, milrinone effectively reduced INa.P under normal condition, hydrogen dioxide-induced INa.P and hypoxia-induced INa.P(Figs. 1, 2). This effect of milrinone is similar to that of TTX. Hydrogen dioxide, a species of reactive oxygen species, whose production is enhanced during ischemia-reperfusion of the heart, is produced as a by-product of oxidative metabolism, in which energy activation and electron reduction are involved (Kourie, 1998). The excessive amount of hydrogen dioxide induces INa.P in ventricular myocytes (Ma et al., 2005). Recent reports show that INa.P induces intracellular sodium overload, which promotes calcium overload via reverse NCX (Haigney et al., 1994; Song et al., 2006; Sossalla et al., 2008), resulting in cell injury (Marsh and Smith, 1991), ventricular electrical instability (Song et al., 2006), ventricular mechanical dysfunction (Belardinellia et al., 2004; Song et al., 2006; Wang et al., 2008) and serious arrhythmia (Thandroyen et al., 1991). In our experiment, hydrogen dioxide prolongs the action potential duration and 2 µM TTX almost eliminates the effect of hydrogen dioxide on the action potential duration (Fig. 4C). This indicates that the action potential duration prolonged by hydrogen dioxide probably results from an increased INa.P induced by hydrogen dioxide. Other studies have shown that increased INa.P not only prolongs action potential duration (Ward and Giles, 1997), but also causes EAD, which may explain, at least in part, the arrhythmogenic effect of hydrogen dioxide on cardiac myocytes (Song et al., 2006). In short, milrinone inhibits prolonged action potential duration associated with hydrogen dioxide-induced INa.P, which is consistent with previous study in guinea pig papillary muscles (Uemura et al., 1999). This shows that milrinone can attenuate exaggeration of transmural dispersion of repolarization and decrease ventricular electrical and mechanical dysfunction. Meanwhile, the inhibitive effect of milrinone on hypoxia or hydrogen dioxide-induced INa.P may reduce the rise in intracellular [Na+] and [Ca2+], which lowers the electrical and mechanical abnormalities associated with such conditions. Furthermore, results of other studies have shown that intracellular sodium overload and calcium overload caused by increased INa.P are more or less associated with pathogenic mechanism of ischemic cardiomyopathy (Saint, 2006), long-QT syndrome 3 (Crotti et al., 2008; Wu et al., 2004) and heart failure (Maltsev et al., 2007; Valdivia et al., 2005). Under these pathological conditions, digitalis poisoning (calcium overload) easily occurs. Thus, as a substitute, milrinone may be beneficial for patients associated with such conditions. Milrinone increases intracellular cAMP by inhibiting type III phosphodiesterase, with the result of a positive inotropic effect on the heart, vasodilatation in the periphery and reduction in ventricular relaxation time (Farah and Frangakis, 1989). Furthermore, in our study, we found while cAMP could inhibit INa.P, H-89 (a highly selective PKA inhibitor) can eliminate the inhibitive effect of milrinone on INa.P. This indicates that milrinone inhibits INa.P via activation of PKA. Another study reported that cAMP could cause a shift in the steady-state inactivation curve of sodium channnel to more negative potentials probably by altering the gating mechanism
211
of the Na+ channel via phosphorylation (caused by activation of PKA) of multiple sites on cardiac Na+ channels (Ono et al., 1989). We conclude that the inhibitive effect of milrinone on INa.P is associated with activation of cAMP-PKA signaling pathway. It has been well documented that milrinone did provide shortterm benefits for heart failure. And a study shows that INa.P is increased in heart failure ventricular cells from a canine pacing model of heart failure and also from explanted failing human hearts (Valdivia et al., 2005). But long-term studies with milrinone showed reduced survival, which is likely to be related to cardiac arrhythimias, possibly resulting from increased intracellular cAMP (Cruickshank, 1993). Despite these data, some physicians have proposed that the regularly scheduled intermittent use of intravenous positive inotropic drugs (e.g., dobutamine or milrinone) in a supervised outpatient setting might be associated with some clinical benefits (Cesario et al., 1998; Marius-Nunez et al., 1996). In summary, milrinone inhibits INa.P under normal condition, hydrogen dioxide- induced INa.P, hypoxia-induced INa.P and the prolonged action potential duration associated with hydrogen dioxide- induced INa.P in ventricular myocytes, which results in attenuated exaggeration of transmural dispersion of repolarization, the electrical instability (formation of EADs and arrhythmia) and mechanical dysfunction. The inhibitive effect of milrinone on hypoxia or hydrogen dioxide-induced INa.P may reduce the rise in intracellular [Na+] and [Ca2+], which lowers the electrical and mechanical abnormalities associated with these conditions. All these effects are associated with the activation of cAMP-PKA signaling pathway. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30670764). References Antzelevitch, C., 2000. Electrical heterogeneity, cardiac arrhythmias, and the sodium channel. Circ. Res. 87, 964–965. Ashamalla, S.M., Navarro, D., Ward, C.A., 2001. Gradient of sodium current across the left ventricular wall of adult rat hearts. J. Physiol. 536, 439–443. Belardinellia, L., Antzelevitch, C., Fraserc, H., 2004. Inhibition of late (sustained/ persistent) sodium current: a potential drug target to reduce intracellular sodiumdependent calcium overload and its detrimental effects on cardiomyocyte function. Eur. Heart J. 6, 13–17. Cesario, D., Clark, J., Maisel, A., 1998. Beneficial effects of intermittent home administration of the inotrope/vasodilator milrinone in patients with end-stage congestive heart failure: a preliminary study. Am. Heart J. 135, 121–129. Chen, E.P., Bittner, H.B., Davis Jr., R.D., Van Trigt III, P., 1997. Milrinone improves pulmonary hemodynamics and right ventricular function in chronic pulmonary hypertension. Ann. Thorac. Surg. 63, 814–821. Crotti, L., Celano, G., Dagradi, F., Schwartz, P.J., 2008. Congenital long QT syndrome. Orphanet. J. Rare Dis. 3, 18. Cruickshank, 1993. Phosphodiesterase III inhibitors: long-term risks and short-term benefits. Cardiovasc. Drugs Ther. 7, 655–660. Farah, A.E., Frangakis, C.J., 1989. Studies on the mechanism of action of the bipyridine milrinone on the heart. Basic Res. Cardiol. 84, 85–103. Friedman, J.E., Haddad, G.G., 1994. Anoxia induces an increase in intracellular sodium in rat central neurons in vitro. Brain Res. 663, 329–334. Ju, Y.K., Saint, D.A., Gage, P.W., 1996. Hypoxia increases persistent sodium current in rat ventricular myocytes. J. Physiol. 497, 337–347. Haigney, M.C., Lakatta, E.G., Stern, M.D., Silverman, H.S., 1994. Sodium channel blockade reduces hypoxic sodium loading and sodium-dependent calcium loading. Circulation 90, 391–399. Hale, S.L., Kloner, R.A., 2006. Ranolazine, an inhibitor of the late sodium channel current, reduces postischemic myocardial dysfunction in the rabbit. J. Cardiovasc. Pharmacol. Ther. 11, 249–255. Hoffman, T.M., Wernovsky, G., Atz, A.M., Bailey, J.M., Akbary, A., Kocsis, J.F., Nelson, D.P., Chang, A.C., Kulik, T.J., Spray, T.L., Wessel, D.L., 2002. Prophylactic intravenous use of milrinone after cardiac operation in pediatrics (PRIMACORP) study. Am. Heart J. 143, 15–21. Hoffman, T.M., Wernovsky, G., Atz, A.M., Kulik, T.J., Nelson, D.P., Chang, A.C., Bailey, J.M., Akbary, A., Kocsis, J.F., Kaczmarek, R., Spray, T.L., Wessel, D.L., 2003. Efficacy and safety of milrinone in preventing low cardiac output syndrome in infants and children after corrective surgery for congenital heart disease. Circulation 107, 996–1002. Honen, B.N., Saint, D.A., 2002. Heterogeneity of the properties of INa in epicardial and endocardial cells of rat ventricle. Clin. Exp. Pharmacol. Physiol. 29, 161–166.
212
J. Zheng et al. / European Journal of Pharmacology 616 (2009) 206–212
Kiyosue, T., Arita, M., 1989. Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes. Circ. Res. 64, 389–397. Kourie, J.I., 1998. Interaction of reactive oxygen species with ion transport mechanisms. Am. J. Physiol. 275, 1–24. Levi, A.J., Dalton, G.R., Hancox, J.C., Mitcheson, J.S., Issberner, J., Bates, J.A., Evans, S.J., Howarth, F.C., Hobai, I.A., Jones, J.V., 1997. Role of intracellular sodium overload in the genesis of cardiac arrhythmias. J. Cardiovasc. Electrophysiol. 8, 700–721. Ma, J.H., Luo, A.T., Zhang, P.H., 2005. Effect of hydrogen peroxide on persistent sodium current in guinea pig ventricular myocytes. Acta Pharmacol. Sin. 26, 828–834. Maltsev, V.A., Silverman, N., Sabbah, H.N., Undrovinas, A.I., 2007. Chronic heart failure slows late sodium current in human and canine ventricular myocytes: implications for repolarization variability. Eur. J. Heart Fail. 9, 219–227. Marius-Nunez, A.L., Heaney, L., Fernandez, R.N., Clark, W.A., Ranganini, A., Silber, E., Denes, P., 1996. Intermittent inotropic therapy in an outpatient setting: a costeffective therapeutic modality in patients with refractory heart failure. Am. Heart J. 132, 805–808. Marsh, J.D., Smith, T.S., 1991. Calcium overload and ischemic myocardial injury. Circulation 83, 709–711. Niemann, J.T., Garner, D., Khaleeli, E., Lewis, R.J., 2003. Milrinone facilitates resuscitation from cardiac arrest and attenuates postresuscitation myocardial dysfunction. Circulation 108, 3031–3035. Ono, K., Kiyosue, T., Arita, M., 1989. Isoproterenol, DBcAMP, and forskolin inhibit cardiac sodium current. Am. J. Physiol. 256, 1131–1137. Saint, D.A., 2006. The role of the persistent Na (+) current during cardiac ischemia and hypoxia. J. Cardiovasc. Electrophysiol. 17, S96–S103. Sakmann, B.F., Spindler, A.J., Bryant, S.M., Linz, K.W., Noble, D., 2000. Distribution of a persistent sodium current across the ventricular wall in guinea pigs. Circ. Res. 87, 910–914. Sasyniuk, B.I., Valois, M., Toy, W., 1989. Recent advances in understanding the mechanisms of drug-induced torsades de pointes arrhythmias. Am. J. Cardiol. 64, 29J–32J. Shaffer, D., Singer, S., Korvick, J., Honig, P., 2002. Concomitant risk factors in reports of torsades de pointes associated with macrolide use: review of the United States Food and Drug Administration Adverse Event Reporting System. Clin. Infect. Dis. 35, 197–200. Shimizu, K., Shintani, Y., Ding, W.G., Matsuura, H., Bamba, T., 2002. Potentiation of slow component of delayed rectifier K(+) current by cGMP via two distinct mechanisms: inhibition of phosphodiesterase 3 and activation of protein kinase G. Br. J. Pharmacol. 137, 127–137. Shipley, J.B., Tolman, D., Hastillo, A., Hess, M.L., 1996. Milrinone: basic and clinical pharmacology and acute and chronic management. Am. J. Med. Sci. 311, 286–291. Simonton, C.A., Chatterjee, K., Cody, R.J., Kubo, S.H., Leonard, D., Daly, P., Rutman, H., 1985. Milrinone in congestive heart failure: acute and chronic hemodynamic and clinical evaluation. J. Am. Coll. Cardiol. 6, 453–459.
Song, Y., Shryock, J.C., Luiz Belardinelli, L., 2008. An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes. Am. J. Physiol. Heart Circ. Physiol. 294, H2031–H2039. Song, Y., Shryock, J.C., Wagner, S., Maier, L.S., Belardinelli, L., 2006. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J. Pharmacol. Exp. Ther. 318, 214–222. Sossalla, S., Wagner, S., Rasenack, E.C., Ruff, H., Weber, S.L., Schöndube, F.A., Tirilomis, T., Tenderich, G., Hasenfuss, G., Belardinelli, L., Maier, L.S., 2008. Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts — role of late sodium current and intracellular ion accumulation. J. Mol. Cell. Cardiol. 45, 32–43. Sugiyama, A., Hashimoto, K., 1999. Can the MAP technique be applied to detect delayed afterdepolarization? Electrophysiologic and pharmacologic evidence. J. Cardiovasc. Pharmacol. 34, 46–52. Sutko, J.L., Kenyon, J.L., Reeves, J.P., 1986. Effects of amrinone and milrinone on calcium influx into the myocardium. Circulation 73, III52–III58. Thandroyen, F.T., Morris, A.C., Hagler, H.K., Ziman, B., Pai, L., Willerson, J.T., Buja, L.M., 1991. Intracellular calcium transients and arrhythmia in isolated heart cells. Circ. Res. 69, 810–819. Uemura, H., Sakamoto, N., Nakaya, H., 1999. Electropharmacological effects of UK-1745, a novel cardiotonic drug, in guinea-pig ventricular myocytes. Eur. J. Pharmacol. 383, 361–371. Valdivia, C.R., Chu, W.W., Pu, J., Foell, J.D., Haworth, R.A., Wolff, M.R., Kamp, T.J., Makielski, J.C., 2005. Increased late sodium current in myocytes from a canine heart failure model and from failing human heart. J. Mol. Cell. Cardiol. 38, 475–483. Wang, L., Lopaschuk, G.D., Clanachan, A.S., 2008. H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation. J. Mol. Cell. Cardiol. 45, 787–795. Ward, C.A., Giles, W.R., 1997. Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. J. Physiol. 500, 631–642. Wu, L., Shryock, J.C., Song, Y., Li, Y., Antzelevitch, C., Belardinelli, L., 2004. Antiarrhythmic effects of ranolazine in a guinea pig in vitro model of long-QT syndrome. J. Pharmacol. Exp. Ther. 310, 599–605. Yunomae, K., Ichisaki, S., Matsuo, J., Nagayama, S., Fukuzaki, K., Nagata, R., Kito, G., 2007. Effects of phosphodiesterase (PDE) inhibitors on human ether-a-go-go related gene (hERG) channel activity. J. Appl. Toxicol. 27, 78–85. Zabel, M., Lichtlen, P.R., Haverich, A., Franz, M.R., 1998. Comparison of ECG variables of dispersion of ventricular repolarization with direct myocardial repolarization measurements in the human heart. J. Cardiovasc. Electrophysiol. 9, 1279–1284. Zygmunt, A.C., Eddlestone, G.T., Thomas, G.P., Nesterenko, V.V., Antzelevitch, C., 2001. Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle. Am. J. Physiol., Heart Circ. Physiol. 281, 689–697.