Stabilization of Kv1.5 channel protein by the inotropic agent olprinone

European Journal of Pharmacology 765 (2015) 488–494

Contents lists available at ScienceDirect

European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Stabilization of Kv1.5 channel protein by the inotropic agent olprinone Ryo Endo a, Yasutaka Kurata b,n, Tomomi Notsu c, Peili Li c, Kumi Morikawa d, Takehito Kondo e, Kazuyoshi Ogura e, Junichiro Miake e, Akio Yoshida c, Yasuaki Shirayoshi c, Haruaki Ninomiya f, Katsumi Higaki g, Masanari Kuwabara h, Kazuhiro Yamamoto e, Yoshimi Inagaki a, Ichiro Hisatome c a

Department of Anesthesiology, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago 683-8503, Japan Department of Physiology, Kanazawa Medical University, Ishikawa 920-0268, Japan c Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, 86 Nishi-cho, Yonago 683-8503, Japan d Center for Promoting Next-Generation Highly Advanced Medicine, Tottori University Hospital, 36-1 Nishi-cho, Yonago 683-8504, Japan e Department of Cardiovascular Medicine, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago 683-8503, Japan f Department of Biological Regulation, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago 683-8503, Japan g Department of Human Genome Science, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago 683-8503, Japan h Department of Cardiology, Toranomon Hospital, Tokyo 105-8470, Japan b

art ic l e i nf o

a b s t r a c t

Article history: Received 31 March 2015 Received in revised form 1 September 2015 Accepted 9 September 2015 Available online 12 September 2015

Olprinone is an inotropic agent that inhibits phosphodiesterase (PDE) III and causes vasodilation. Olprinone has been shown to be less proarrhythmic and possibly affect expression of functional Kv1.5 channels that confer the ultra-rapid delayed-rectifier K þ channel current (IKur) responsible for action potential repolarization. To reveal involvement of Kv1.5 channels in the less arrhythmic effect of olprinone, we examined effects of the agent on the stability of Kv1.5 channel proteins expressed in COS7 cells. Olprinone at 30–1000 nM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that olprinone delayed degradation of Kv1.5 channels. Olprinone increased the immunofluorescent signal of Kv1.5 channels in the endoplasmic reticulum (ER) and Golgi apparatus as well as on the cell surface. Kv1.5-mediated membrane currents, measured as 4-aminopyridine-sensitive currents, were increased by olprinone without changes in their activation kinetics. A protein transporter inhibitor, colchicine, abolished the olprinone-induced increase of Kv.1.5-mediated currents. The action of olprinone was inhibited by 4-aminopyridine, and was not mimicked by the application of 8-Bromo-cAMP. Taken together, we conclude that olprinone stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, and thereby increases the density of Kv1.5 channels on the cell membrane. The enhancement of Kv1.5 currents could underlie less arrhythmogenicity of olprinone. & 2015 Elsevier B.V. All rights reserved.

Keywords: Olprinone Kv1.5 channel Chemical chaperone

1. Introduction Olprinone (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), an analog of milrinone, exerts positive inotropic and vasodilative actions. It is a phosphodiesterase (PDE) III inhibitor clinically used in the treatment of acute heart failure (Kanaya et al., 2001). In anesthetized dogs, it increased cardiac contractility, decreased systemic vascular resistance with little reduction in the mean aortic pressure and heart rate (Ogawa et al., 1989; Ohhara et al., 1989;

n Correspondence to: Department of Physiology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. E-mail address: [email protected] (Y. Kurata).

http://dx.doi.org/10.1016/j.ejphar.2015.09.013 0014-2999/& 2015 Elsevier B.V. All rights reserved.

Satoh and Endoh, 1990), and improved myocardial mechanical efficiencies without increasing oxygen consumption (Mizushige et al., 2002). Clinical studies have showed that olprinone has advantages of facilitating release from artificial cardio-pulmonary devices and improving hemodynamics and peripheral circulation after release from artificial cardio-pulmonary devices (Sha et al., 2003). While other phosphodiesterase III inhibitors such as amlinone and milrinone also showed inotropic and vasodilative actions, they were less effective than olprinone (Ohhara et al., 1992). Olprinone is known to have a cardioprotective action as well: repetitive treatment with olprinone prior to myocardial ischemia led to a cardioprotective action in heart failure after myocardial infarction of rats (Tosaka et al., 2007, 2011; Matsumoto et al., 2009), which may be due to the activation of the cAMP/PKA pathway via inhibition of PDE III.

R. Endo et al. / European Journal of Pharmacology 765 (2015) 488–494

Phosphorylation of L-type Ca2 þ channels enhances Ca2 þ influx and Ca2 þ -induced Ca2 þ -release from the sarcoplasmic reticulum, leading to Ca2 þ overload and lethal arrhythmias in failing hearts (Sipido et al., 2000; Naccarelli and Goldstein, 1989). Ca2 þ overload induced by PDE III inhibitors is attributed to increased Ca2 þ entry via enhancement of the L-type Ca2 þ channel current (ICaL) along with prolonged action potential duration (APD). The dual pharmacophore ATI22-107 that causes both PDE III inhibition and Ca2 þ channel block has showed a positive inotropic action on hearts without elevating intracellular Ca2 þ concentrations (Jung et al., 2005). Alternatively, the PDE III inhibitors that shorten APD by activating K þ channels might also exert a positive inotropic effect without inducing Ca2 þ overload. Kv1.5 is a member of the voltage-gated K þ channel superfamily that confers the ultra-rapid delayed-rectifier K þ channel current (IKur) (Nattel et al., 1999), which shows slow time-dependent inactivation and is blocked by 4-aminopyridine (4AP). Kv1.5 channels predominantly expressed in atrial myocytes contribute to action potential (AP) repolarization and strongly affect APD. It has recently been reported that Kv1.5 channels are expressed in ventricular myocytes as well and regulate their AP repolarization (Cheng et al., 2011). Recent studies also indicated that protein and mRNA levels of Kv1.5 were significantly reduced in the hearts of dilated cardiomyopathy (DCM) and lethal arrhythmia model mice (Suzuki et al., 2012). These model mice have prolonged ventricular APDs and long QT intervals in electrocardiogram. Overexpression of Kv1.5 using adenoviral vectors normalized ventricular APDs and QT intervals in the model mice, suggesting the effectiveness of enhanced Kv1.5 expression in counteracting arrhythmogenicity. In the present study, we examined effects of chronic treatment with olprinone on the stability and expression of Kv1.5 channels by molecular biological and electrophysiological approaches. Olprinone stabilized the Kv1.5 channel and enhanced its expression on the plasma membrane, which may underlie the less arrhythmogenic effect of the agent.

2. Materials and methods 2.1. Transient expression of Kv1.5-FLAG proteins in cultured cells The expression construct pRC/Kv1.5-FLAG was engineered by ligating an oligonucleotide encoding a FLAG epitope to the carboxy terminus of rat Kv1.5 cDNA. African green monkey kidney fibroblast (COS7) cells were used for transient expression of Kv1.5-FLAG and to study effects of olprinone on Kv1.5-FLAG protein stability. In brief, cells were maintained in Dulbecco's modified Eagle medium (Gibco BRL, USA)/10% fetal bovine serum (Gibco BRL) at 37 °C in a 5% CO2 incubator, and transfected with pRC/Kv1.5-FLAG together with the plasmid for enhanced green fluorescent protein (pEGFP) using lipofectamine (Gibco BRL). Forty-eight hours after transfection, cells were subjected to assays. Colchicine was added to the culture medium at 36 h after transfection in order to inhibit ADPribosylation factor-dependent and microtubule-dependent protein transport from the endoplasmic reticulum (ER) and Golgi apparatus to the plasma membrane (Kato et al., 2005). Colchicine was dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the culture medium was equal to or less than 0.01% (v/v). 2.2. Western blotting Cells were scraped into lysis buffer [phosphate buffer saline (PBS)/1% NP40 (Nonidet P-40: polyoxyethylene(9)octyiphenyl ether), 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), 10 μg/ml aprotinin, 10 μg/ml leupeptine, 10 μg/ml pepstain

489

and 1 mM phenylmethylsulfonylfluoride], lysed by sonication, and then centrifuged to remove insoluble materials. Protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Biotechnology, Rockford, IL, USA). An aliquot of 10 μg protein was subjected to sodium dodecylsulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were probed with antibodies against FLAG (1:1000, Cosmo Bio), α-actin (1:5000, Oncogene) and green fluorescent protein (GFP) (1:4000, Molecular Probes), and developed using an electrochemiluminescence (ECL) Western blotting detection system. 2.3. Degradation assays To analyze the degradation kinetics of Kv1.5-FLAG, COS7 cells were seeded in 6-well plates and transfected with pRC/Kv1.5FLAG. Protein lysates were prepared at 0, 2, 6, 12, 24 and 48 h after the addition of cycloheximide (60 μg/ml). Cells were washed once with PBS and lysed in the lysis buffer. The lysates were centrifuged at 20,000 g for 30 min at 4 °C to obtain soluble extracts. Equal amounts of the protein were analyzed by immunoblotting. Band intensity was quantified using an NIH image software. The decay rate constant (k) was estimated by fitting the first-order decay curve of the form y¼e  kt to the time-dependent band intensity data, using SigmaPlot (Jandel Scientific, USA). The half-life (t1/2) of the protein was calculated using the formula t1/2 ¼0.693/k (Kato et al., 2005). 2.4. Immunofluorescence COS7 cells were transfected with pRC/Kv1.5-FLAG, together with the plasmid for the ER-targeted GFP (AcGFP-ER), Golgi-targeted GFP (AcGFP-Golgi), plasma membrane-targeted GFP (AcGFPMem) or endosome-enhanced GFP (endosome-EGFP) (Clontech, USA). Fixed cells were stained for anti-FLAG antibody as described previously (Kato et al., 2005) using Texas Red-conjugated antimouse IgG as the secondary antibody. The distributions of Kv1.5FLAG, AcGFP-ER, AcGFP-Golgi, AcGFP-Mem and endosome-EGFP were examined using a Zeiss LSM700 confocal microscope illuminated with a krypton/argon laser (Deutschland, Germany). Excitation and emission filters were used at wavelengths of 540 and 480 nm. The background fluorescence was normalized to that of negative control. Fluorescent images were prepared from data files and quantified with Zeiss ZEN 2009 software and Adobe Photoshop software (San Jose, CA, USA). Linear adjustments in brightness and/or contrast were applied to the entire image when necessary. All immunofluorescence assays were performed in four independent experiments. 2.5. Electrophysiological recordings COS7 cells were co-transfected with pRC/Kv1.5-FLAG together with pEGFP. Transfected cells were visualized by EGFP fluorescence and subjected to whole-cell patch clamp experiments to measure Kv1.5-mediated membrane currents. Currents were measured as previously described by Kato et al. (2005). Briefly, currents were elicited every 6 s by 500-ms test pulses ranging from  60 to þ80 mV (in 10 mV increments) with a holding potential of  60 mV. Kv1.5 currents were measured as 4AP-sensitive currents, i.e., the differences between the currents consecutively recorded in the absence and presence of 1 mM 4AP (Kato et al., 2005). To determine the voltage-dependent activation kinetics of Kv1.5 currents, we measured Kv1.5 currents using 100-ms conditioning pulses from  60 to þ 80 mV (in a 10 mV increment)

490

R. Endo et al. / European Journal of Pharmacology 765 (2015) 488–494

followed by a fixed repolarizing test pulse of  30 mV. The peak of tail currents during the repolarizing pulse was determined for each conditioning pulse potential and normalized to the value at þ80 mV. Half-maximal voltage (Eh) and slope factor (s) of the voltage-dependent current activation were determined by fitting the data with a Boltzmann equation,

y = 1/{1 + exp[ − (V − Eh )/s]} ,

(1)

0h

2h 6h 12h 24h 48h

Control Olprinone (100 nM)

All data are expressed as the mean 7S.E.M. For statistical analyses, Mann–Whitney's U test, two-way repeated measures analysis of variance (ANOVA) and Kruskal–Wallis test were used, with Po 0.05 being considered statistically significant. When a significant difference was observed with the repeated measures ANOVA or Kruskal–Wallis test, paired comparisons were made within a group and also between groups by using the Dunnett post-hoc test.

Kv1.5-FLAG density

2.6. Statistical analysis

(normalized to time 0 value)

where y denotes the steady-state open probability of Kv1.5 channels (activation gate) at a membrane potential V.

1.2

Control (n=4) Olprinone (n=4)

1.0 0.8

*

0.6

*

0.4

* 0.2 0.0

3. Results 3.1. Concentration-dependent effects of olprinone on the stability of Kv1.5 channel proteins Effects of olprinone on protein levels of Kv1.5-FLAG were examined in transfected COS7 cells. Fig. 1A shows a representative western blot of Kv1.5-FLAG proteins. Olprinone increased the level of Kv1.5-FLAG proteins in a concentration-dependent manner with the maximum effect attained at 300 nM, as summarized in Fig. 1B.

0

10

20

30

40

50

Time (hrs) Fig. 2. Effects of olprinone on the stability of Kv1.5-FLAG proteins expressed in COS7 cells. (A) Representative immunoblots in control and in the presence of 100 nM olprinone. Cells were treated with cycloheximide (60 μg/ml) and chased for the indicated times. (B) Time-dependent changes in the density of Kv1.5-FLAG proteins with fitted single exponential decay lines (n ¼4). Protein densities were normalized to the value at time 0. Differences were tested for statistical significance by two-way repeated measures ANOVA and Dunnett post-hoc test: nPo 0.05 (vs. Control).

Olprinone (nM) C

30

100

300

1000

Kv1.5-FLAG β -actin

GFP

Effects of olprinone on the stability of Kv1.5-FLAG proteins were further examined using the cyclohexamide chase assay. Olprinone at 100 nM significantly slowed the degradation of Kv1.5-FLAG proteins (Fig. 2): the t1/2 values in the absence and presence of olprinone was 6.6 7 1.0 h (n¼ 4) and 11.871.5 h (n ¼4; P o0.05), respectively.

Normalized ratio of Kv1.5-FLAG tot β actin (%)

3.2. Effects of olprinone on the intracellular localization of Kv1.5 channel proteins

150

*

*

30

100

*

*

300

1000

100 50 0

C

Olprinone (nM) Fig. 1. Concentration-dependent effects of 30–1000 nM olprinone on the protein level of Kv1.5-FLAG expressed in COS7 cells. (A) Representative Western blots. Cells were treated with different concentrations of olprinone for 12 h and cell extracts were subjected to Western blotting with indicated antibodies. Control data obtained without olprinone treatment are labeled as C. (B) Quantification of the protein levels of Kv1.5-FLAG for individual concentrations of the agent (n¼ 8 each). Each bar represents the band density (mean 7 S.E.M.) normalized to the control band density in the absence of olprinone (C). Differences were tested for statistical significance by Kruskal–Wallis test and Dunnett post-hoc test: nP o0.05 (vs. C).

Intracellular localization of Kv1.5-FLAG proteins was examined by immuno- fluorescence. Signals of Kv1.5-FLAG proteins were detected on the plasma membrane, ER and Golgi apparatus as shown in Fig. 3A by their co-localization with AcGFP-Mem (panels a–c), AcGFP-ER (panels d–f), and AcGFP-Golgi (panels g–i), respectively; olprinone at 100 nM remarkably increased the signals of Kv1.5-FLAG proteins on the plasma membrane (panels a’–c’), ER (panels d’–f’) and Golgi apparatus (panels g’–i’), as summarized in Fig. 3B. 3.3. Effects of olprinone on the expression of functional Kv1.5 channels To examine whether olprinone increases the density of functional Kv1.5-FLAG channels on the cell surface, we measured Kv1.5-mediated membrane currents. In cells expressing Kv1.5FLAG, depolarizing test pulses elicited outward currents, which were almost completely blocked by 1 mM 4AP. Treatment with olprinone at 100 nM for 12 h increased the amplitude of expressed Kv1.5-FLAG currents (Fig. 4A). As summarized in Fig. 4B, olprinone

R. Endo et al. / European Journal of Pharmacology 765 (2015) 488–494

491

20μm

Kv

GFP

merged

merged

Kv

GFP

PM a

b

c

a’

b’

c’

d

e

f

d’

e’

f’

g

h

i

g'

h’

i’

ER

Golgi

Kv1.5-FLAG signal intensity

Control

Olprinone (100 nM)

*

0.30 0.25

*

*

0.20 0.15 0.10 0.05 0.00

Olprinone (100 nM)

ER

Golgi

PM

Fig. 3. Effects of olprinone on intracellular localization of Kv1.5-FLAG proteins determined by immunofluorescence in COS7 cells. (A) Representative images obtained with a confocal microscope from cells expressing Kv1.5-FLAG (Kv) together with AcGFP-Mem (PM), AcGFP-ER (ER), or AcGFP-Golgi (Gogi). GFP (for PM, ER and Golgi), Kv1.5 and merged images (merge) are shown. Cells were treated with 100 nM olprinone or vehicle (Control) for 12 h and stained with the anti-FLAG antibody. Bar: 20 μm. (B) Quantification of anti-FLAG immunoreactivity. Each bar represents the ratio of Alexa 555 fluorescence intensity of Kv1.5-FLAG proteins to that of AcGFP-ER, AcGFP-Golgi or AcGFP-Mem. Signals for the Kv1.5-FLAG protein and markers were quantified with the software for Zeiss LSM700. Differences were tested for statistical significance by Mann–Whitney's U test (n¼ 8): nP o 0.05 (vs. each Control).

at 100 nM significantly augmented Kv1.5-FLAG currents at the membrane potentials ranging from þ 20 to þ80 mV without affecting the threshold potential of the current activation. Fig. 4C shows the representative original traces of tail currents recorded for determination of the steady-state voltage-dependent activation curve for Kv1.5-FLAG currents in transfected cells in the absence and presence of olprinone (100 nM). The activation curves were constructed by measuring peak tail currents at a repolarizing potential of  30 mV, as described in the materials and methods section. A 12-h treatment with 100 nM olprinone did not cause any significant change in the activation curve: in control, Eh ¼  7.1 73.3 mV and s¼5.0 70.8; with olprinone, Eh ¼  6.5 7 3.1 mV and s ¼5.2 70.5 (Fig. 4D). Olprinone did not significantly alter the time course of current activation during depolarizing pulses or current decay during repolarizing pulses. Thus, olprinone increased the density of Kv1.5-FLAG currents without changing the voltage-dependent activation kinetics. We further investigated effects of a protein transport inhibitor, colchicines, on Kv1.5-FLAG currents. Fig. 5A shows representative traces of Kv1.5-FLAG currents recorded from cells treated with or without colchicine. Treatment with this agent at 5 μM for 12 h

significantly decreased the basal level of Kv1.5-FLAG currents and minimized the olprinone-induced increase of the currents as summarized in Fig. 5B. Colchicine did not cause any changes in the currents when applied immediately before the recording (data not shown). These findings were reproduced by another protein transporter inhibitor, brefeldin (data not shown). 3.4. A potential action of olprinone as a chemical chaperon Ion channel inhibitors stabilize the structure of proteins, and have an activity of the chemical chaperon: e.g., the Kv1.5 channel blocker 4AP has been reported to act as a chemical chaperone to stabilize Kv1.5 proteins (Koshida et al., 2009; Suzuki et al., 2012). Fig. 6A shows the levels of Kv1.5-FLAG proteins in cells pretreated with 4AP alone or together with olprinone. 4AP at 100 μM significantly increased the level of Kv1.5-FLAG proteins, indicating its chemical chaperone action. Olprinone at 100 nM failed to further increase the Kv1.5-FLAG level in the presence of 100 μM 4AP. As summarized in Fig. 6B, 4AP abolished the enhancement of Kv1.5FLAG protein expression by olprinone. Thus, the stabilizing effect of olprinone on Kv1.5-FLAG channel proteins was inhibited by the

492

R. Endo et al. / European Journal of Pharmacology 765 (2015) 488–494

Control pretreatment

Olprinone

subtraction

4AP

Control

Olprinone

1 nA 1 nA 100 ms

100 ms

Normalized Currents

4AP-sensitive Currents (pA/pF)

1.2 1.2

100 80

Control (n=11) Olprinone (n=11)

** **

60

**

**

40 20 0 -80 -60 -40 -20 0

20 40 60 80 100

Control (n=5) Olprinone (n=5)

1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 -80 -60 -40 -20 -80 -60 -40 -20

00

20 20

40 40

60 60

80 100 80 100

Conditioning Potentials (mV)

Test potential (mV)

Fig. 4. Effects of chronic treatment with olprinone on membrane currents in COS7 cells expressing Kv1.5-FLAG. (A) Representative current traces recorded from cells transfected with Kv1.5-FLAG plasmid for 48 h in the absence (top) and presence (bottom) of 100 nM olprinone. The currents were elicited by 300-ms test pulses ranging from  60 to þ 80 mV (in 20 mV increments). The holding potential was  60 mV. The currents were recorded in the absence (left) and presence (middle) of 1 mM 4AP; the 4APsensitive currents (right) were obtained by subtracting the currents recorded in the presence of 1 mM 4AP from those recorded in its absence. (B) Effect on the voltagedependent activation of 4AP-sensitive currents. Peak amplitudes of the currents determined with (closed circles) and without (open circles) olprinone treatment are presented as means 7 S.E.M. (pA/pF). Statistical significance of the differences between olprinone and control group was tested by two-way repeated measures ANOVA and Dunnett post-hoc test (n¼ 11): nnPo 0.01 (vs. Control). (C) Representative current traces recorded by the double pulse protocol to determine the steady-state activation curve for 4AP-sensitive Kv1.5-FLAG currents after 12-h treatment with vehicle (left) or 100 nM olprinone (right). Tail currents were elicited by 100-ms repolarizing test pulses to  30 mV, following 100-ms conditioning pulses ranging from  60 to þ80 mV (in 10 mV increments). (D) Steady-state activation curves of Kv1.5-FLAG currents with (closed circles) and without (open circles) olprinone treatment. Peak amplitudes of the tail currents were normalized to the maximum amplitude attained at depolarized potentials, and plotted as functions of the conditioning potentials. The curves are the fits to the averaged data with Eq. (1).

chemical chaperone that directly binds to the Kv1.5 protein. To determine whether an increase in intracellular cAMP could influence the protein level of Kv1.5 or not, we further examined effects of 8-bromo-cAMP on Kv1.5-FLAG protein levels. 8-bromocAMP at 20 μM did not significantly increase the protein level of Kv1.5-FLAG, nor did it inhibit the increasing effect of 100 nM olprinone on the Kv1.5 protein level (Fig. 7). Thus, the stabilizing effect of olprinone on Kv1.5-FLAG channel proteins was not

Control

+Colchicine

Olprinone

+Colchicine

attributable to its action of elevating intracellular cAMP.

4. Discussion In the present study, we found that (1) olprinone slowed the degradation of the Kv1.5-FLAG protein and increased its level in a concentration-dependent manner; (2) olprinone enhanced the

1nA 100ms Fig. 5. Effects of the protein transport blocker colchicine on the olprinone-induced increase of Kv1.5-FLAG currents. (A) Representative original traces of Kv1.5-FLAG currents recorded from cells of the control and 100 nM olprinone-treated groups. Transfected cells were treated with 5 μM colchicine for 12 h before determination of 4AP-sensitive Kv1.5-FLAG currents. (B) Averaged peak amplitudes of Kv1.5-FLAG currents at þ 80 mV recorded from the control and olprinone and/or colchicines-treated cells. Each bar represents the mean 7 S.E.M. (n ¼15). nPo 0.05 (by two-way repeated measures ANOVA and Dunnett post-hoc test).

R. Endo et al. / European Journal of Pharmacology 765 (2015) 488–494

Control

+4AP

+4AP +Olprinone

Control

cAMP

Olprinone

Kv1.5-FLAG

Kv1.5-FLAG

β-actin

β -actin

GFP

GFP N.S.

2.5

*

2.0 1.5 1.0 0.5 0.0 Control

*

2.5

+4AP

+4AP +Olprinone (100 nM)

Fig. 6. Effects of the Kv1.5 channel blocker 4AP on the olprinone-induced increase of Kv1.5-FLAG expression. Shown are representative Western blots with indicated antibodies (A) and intensities of Kv1.5-FLAG proteins normalized to those of β-actin (B). Cells were treated with vehicle (Control) or 100 nM olprinone following pretreatment with 100 μM 4AP (n ¼4). Each bar represents the mean 7S.E.M. nP o 0.05 (by Kruskal–Wallis test and Dunnett post-hoc test).

signals of Kv1.5-FLAG proteins in the ER and Golgi as well as on the plasma membrane, increasing the Kv1.5-FLAG current without any changes in its activation kinetics; (3) colchicine and 4AP abolished the action of olprinone to increase the levels of Kv1.5-FLAG proteins and currents; and (4) 8-bromo-cAMP did not increase Kv1.5FLAG expression or inhibit the olprinone effect. Olprinone has been reported to cause an increase of contractile force with small increases in automaticity in isolated rat atrial muscles (Ogawa et al., 1989). It enhanced the contraction of isolated papillary and right ventricular muscles with remarkable prolongation of APDs in a high-potassium solution (Ogawa et al., 1989). The inotropic response of papillary muscles to isoproterenol was potentiated by pretreatment with olprinone at a minimally-effective inotropic concentration of 30 nM (Ogawa et al., 1989). These actions are mediated by increased cAMP through inhibition of PDE III (Ogawa et al., 1989; Satoh and Endoh, 1990). Olprinone also shortened APDs of ventricular myocytes and accelerated pacemaker depolarization in sinoatrial node cells (Ohhara et al., 1992). In the present study, olprinone increased immunofluorescent signals of Kv1.5-FLAG proteins on the plasma membrane and Kv1.5 currents without changes in their activation kinetics, which is a possible mechanism of the APD shortening. The main site of this action appeared to be the ER: olprinone increased the signals of Kv1.5FLAG proteins in the ER and Golgi apparatus, suggesting that the enhancement of Kv1.5-FLAG expression by olprinone is secondary to stabilization of Kv1.5-FLAG proteins in the ER. This notion is consistent with the effect of colchicine, which probably inhibited intracellular transport of mature Kv1.5-FLAG proteins from the ER/ Golgi apparatus to the cell surface and abolished the enhancing effects of olprinone on Kv1.5-FLAG protein expressions. Adrenergic activation is well known to modulate the electrical properties of K þ channel currents (Yue et al., 1999). Olprinone is reported to shorten APDs of cardiomyocytes, suggesting that it could enhance outward K þ currents via accumulation of cAMP (Mason et al., 2002). Several studies reported that IKur was increased by PKA in response to β-adrenergic activation; IKur was enhanced by

Ratio of Kv1.5-FLAG to β -actin (%)

Ratio of Kv1.5-FLAG to β- actin (%)

493

2.0 1.5 1.0 0.5 0.0 Control

cAMP (20 μM)

Olprinone (100 nM)

Fig. 7. Comparison of the effects of pretreatment with bromo-cAMP and olprinone on Kv1.5-FLAG expression. (A) Representative Western blots in control (left), in the presence of 20 μM 8-bromo-cAMP alone (middle), and in the presence of both 20 μM 8-bromo-cAMP and 100 nM olprinone (right). (B) Normalized intensities of Kv1.5-FLAG proteins. Cells were treated with vehicle (Control), 20 μM 8-bromocAMP alone, or both 20 μM 8-bromo-cAMP and 100 nM olprinone (n¼4 each). Each bar represents the mean 7 S.E.M. nP o0.05 (vs. Control by Kruskal–Wallis test and Dunnett post-hoc test).

isoproterenol and forskolin as well as 8-bromo-cAMP, and was inhibited by PKA inhibitors (Yue et al., 1999; Mason et al., 2002). Thus, the olprinone-induced stabilization of Kv1.5-FLAG proteins and enhancement of Kv1.5-FLAG currents might be due to phosphorylation of Kv1.5 proteins by activated PKA. However, 8-bromo-cAMP failed to stabilize Kv1.5-FLAG proteins. The Kv1.5 channel blocker 4AP, which induces conformational changes of Kv1.5 proteins and enhances their expression, abolished the increase of Kv1.5-FLAG protein expression by olprinone. This finding suggests that olprinone stabilizes Kv1.5 proteins via the direct binding to the protein and subsequent alterations in its conformation. We found that the chronic treatment with olprinone at 30– 1000 nM could stabilize Kv1.5-FLAG proteins. The minimum effective concentration of olprinone (30 nM) is less than its therapeutic plasma concentrations of around 100 nM (Kurokawa et al., 2008). The clinical relevance of the effects of olprinone on Kv1.5 expression is not clear; however, olprinone did not cause prominent tachycardia or arrhythmias (Adachi and Tanaka, 1997), which may be explained by enhancement of the Kv1.5-mediated current IKur. Kv1.5 is known to be expressed predominantly in atrial myocytes, where it significantly contributes to AP repolarization. It has been reported that a reduction of Kv1.5 currents caused by genetic mutation leads to automaticity of the atrial muscle (Olson et al., 2006). Thus, increasing the Kv1.5 channel density by olprinone would suppress the automaticity of atrial muscles. The augmentation of IKur resulted in a shortening of APDs and a reduction of Ca2 þ load in the atrium (Fujiki et al., 2004), and might also suppress the automaticity of atrial muscles (Miyaji et al., 2007). Kv1.5 has recently been reported to be expressed in ventricular myocytes as well (Kato et al., 2005). Recent studies also

494

R. Endo et al. / European Journal of Pharmacology 765 (2015) 488–494

indicated that protein and mRNA levels of Kv1.5 were significantly reduced in DCM model mouse hearts, which might be related to lethal arrhythmias in this model mouse (Suzuki et al., 2012). Since overexpression of Kv1.5 using adenoviral vectors normalized the ventricular APD and suppressed ventricular arrhythmias in DCM mice with prolonged QT intervals (Suzuki et al., 2012), stabilization of Kv1.5 proteins by olprinone may lead to suppression of ventricular arrhythmias in the failing hearts with prolonged QT intervals. Several studies reported that olprinone exerts cardioprotective effects via several mechanisms such as modification of phosphatidylinositol-3-OH kinase-Akt and a mitochondrial permeability transition pore during early reperfusion (Tosaka et al., 2007, 2011; Matsumoto et al., 2009). The stabilization of Kv1.5 channel proteins might be a novel mechanism underlying the cardioprotective actions of olprinone.

Disclosures None.

Source of support This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan (No. 25670110) to IH.

References Adachi, H., Tanaka, H., 1997. Effects of a new cardiotonic phosphodiesterase III inhibitor, olprinone, on cardiohemodynamics and plasma hormones in conscious pigs with heart failure. J. Cardiovasc. Pharmacol. 29, 763–771. Cheng, L., Yung, A., Covarrubias, M., Radice, G.L., 2011. Cortactin is required for N-cadherin regulation of Kv1.5 channel function. J. Biol. Chem. 286, 20478–20489. Fujiki, A., Sakabe, M., Nishida, K., Sugao, M., Tsuneda, T., Iwamoto, J., Mizumaki, K., Inoue, H., 2004. Drug-induced changes in fibrillation cycle length and organization index can predict chemical cardioversion of long-lasting atrial fibrillation with bepridil alone or in combination with aprindine. Circ. J. 68, 1139–1145. Jung, A.S., Quaile, M.P., Mills, G.D., Bednarik, D.P., Houser, S.R., Margulies, K.B., 2005. Pharmacological effects of ATI22-107 [2-(2-{2-[2-chloro-4- (6-oxo-1,4,5,6-tetrahydro-pyridazin-3-yl)-phenoxy]-acetylami no}-ethoxymethyl)- 4-(2-chlorophenyl)-6-methyl-1,4-dihydro-pyridine-3,5-dicarboxy lic acid dimethyl ester)], a novel dual pharmacophore, on myocyte calcium cycling and contractility. J. Pharmacol. Exp. Ther. 312, 517–524. Kanaya, N., Murray, P.A., Damron, D.S., 2001. Propofol increases myofilament Ca2 þ sensitivity and intracellular pH via activation of Na þ –H þ exchange in rat ventricular myocytes. Anesthesiology 94, 1096–1104. Kato, M., Ogura, K., Miake, J., Sasaki, N., Taniguchi, S., Igawa, O., Yoshida, A., Hoshikawa, Y., Murata, M., Nanba, E., Kurata, Y., Kawata, Y., Ninomiya, H., Morisaki, T., Kitakaze, M., Hisatome, I., 2005. Evidence for proteasomal degradation of Kv1.5 channel protein. Biochem. Biophys. Res. Commun. 337, 343–348. Koshida, S., Kurata, Y., Notsu, T., Hirota, Y., Kuang, T.Y., Li, P., Bahrudin, U., Harada, S., Miake, J., Yamamoto, Y., Hoshikawa, Y., Igawa, O., Higaki, K., Soma, M., Yoshida, A., Ninomiya, H., Shiota, G., Shirayoshi, Y., Hisatome, I., 2009. Stabilizing effects of eicosapentaenoic acid on Kv1.5 channel protein expressed in mammalian cells. Eur. J. Pharmacol. 604, 93–102. Kurokawa, H., Matsunaga, A., Tanaka, H., Hamada, H., Kawamoto, M., Yuge, O., 2008.

Clinically relevant concentrations of olprinone reverse attenuating effect of propofol on isoproterenol-induced cyclic adenosine monophosphate accumulation in cardiomyocytes. Hiroshima J. Med. Sci. 57, 1–6. Mason, H.S., Latten, M.J., Godoy, L.D., Horowitz, B., Kenyon, J.L., 2002. Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton. Mol. Pharmacol. 61, 285–293. Matsumoto, S., Cho, S., Tosaka, S., Ureshino, H., Maekawa, T., Hara, T., Sumikawa, K., 2009. Pharmacological preconditioning in type 2 diabetic rat hearts: the roles of mitochondrial ATP-sensitive potassium channels and the phosphatidylinositol 3-kinase-Akt pathway. Cardiovasc. Drugs Ther. 23, 263–270. Miyaji, K., Tada, H., Fukushima, Kusano, K., Hashimoto, T., Kaseno, K., Hiramatsu, S., Tadokoro, K., Naito, S., Nakamura, K., Oshima, S., Taniguchi, K., Ohe, T., 2007. Efficacy and safety of the additional bepridil treatment in patients with atrial fibrillation refractory to class I antiarrhythmic drugs. Circ. J. 71, 1250–1257. Mizushige, K., Ueda, T., Yukiiri, K., Suzuki, H., 2002. Olprinone: a phosphodiesterase III inhibitor with positive inotropic and vasodilator effects. Cardiovasc. Drug Rev. 20, 163–174. Naccarelli, G.V., Goldstein, R.A., 1989. Electrophysiology of phosphodiesterase inhibitors. Am. J. Cardiol. 63, 35A–40A. Nattel, S., Yue, L., Wang, Z., 1999. Cardiac ultrarapid delayed rectifiers: a novel potassium current family of functional similarity and molecular diversity. Cell. Physiol. Biochem. 9, 217–226. Ogawa, T., Ohhara, H., Tsunoda, H., Kuroki, J., Shoji, T., 1989. Cardiovascular effects of the new cardiotonic agent 1,2-dihydro-6-methyl-2-oxo-5- (imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate. 1st communication: studies on isolated guinea pig cardiac muscles. Arzneimittelforschung 39, 33–37. Ohhara, H., Ogawa, T., Takeda, M., Katoh, H., Daiku, Y., Igarashi, T., 1989. Cardiovascular effects of the new cardiotonic agent 1,2-dihydro-6-methyl-2-oxo-5(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate. 2nd communication: studies in dogs. Arzneimittelforschung 39, 38–45. Ohhara, H., Sawada, K., Ogawa, T., Takeda, M., Igarashi, T., 1992. [Effects of the new cardiotonic agent loprinone hydrochloride (E-1020) on left ventricular diameter in normal and experimental heart failure dogs and its action potential characteristics in isolated guinea pig cardiac muscles and sinus nodes]. Nihon Yakurigaku Zasshi 99, 421–433. Olson, T.M., Alekseev, A.E., Liu, X.K., Park, S., Zingman, L.V., Bienengraeber, M., Sattiraju, S., Ballew, J.D., Jahangir, A., Terzic, A., 2006. Kv1.5 channelopathy due to KCNA5 loss-of-function mutation causes human atrial fibrillation. Hum. Mol. Genet. 15, 2185–2191. Satoh, H., Endoh, M., 1990. Effects of a new cardiotonic agent 1,2-dihydro-6- methyl-2-oxo-5-[imidazo (1,2-a) pyridin-6-yl]-3-pyridine carbonitrile hydrochloride monohydrate (E-1020) on contractile force and cyclic AMP metabolism in canine ventricular muscle. Jpn. J. Pharmacol. 52, 215–224. Sha, K., Iwata, M., Mori, M., Motozu, Y., Yonemoto, N., Kitaguchi, K., Hirai, K., Furuya, H., 2003. [Evaluation of the different doses of olprinone for continuous infusion after cardiopulmonary bypass for coronary artery bypass surgery]. Masui 52, 621–625. Sipido, K.R., Volders, P.G., de Groot, S.H., Verdonck, F., Van de Werf, F., Wellens, H.J., Vos, M.A., 2000. Enhanced Ca2 þ release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis. Circulation 102, 2137–2144. Suzuki, S., Kurata, Y., Li, P., Notsu, T., Hasegawa, A., Ikeda, N., Kato, M., Miake, J., Sakata, S., Shiota, G., Yoshida, A., Ninomiya, H., Higaki, K., Yamamoto, K., Shirayoshi, Y., Hisatome, I., 2012. Stabilization of Kv1.5 channel protein by bepridil through its action as a chemical chaperone. Eur. J. Pharmacol. 696, 28–34. Suzuki, T., Shioya, T., Murayama, T., Sugihara, M., Odagiri, F., Nakazato, Y., Nishizawa, H., Chugun, A., Sakurai, T., Daida, H., Morimoto, S., Kurebayashi, N., 2012. Multistep ion channel remodeling and lethal arrhythmia precede heart failure in a mouse model of inherited dilated cardiomyopathy. PLoS One 7, e35353. Tosaka, S., Makita, T., Tosaka, R., Maekawa, T., Cho, S., Hara, T., Ureshino, H., Sumikawa, K., 2007. Cardioprotection induced by olprinone, a phosphodiesterase III inhibitor, involves phosphatidylinositol-3-OH kinase-Akt and a mitochondrial permeability transition pore during early reperfusion. J. Anesth. 21, 176–180. Tosaka, S., Tosaka, R., Matsumoto, S., Maekawa, T., Cho, S., Sumikawa, K., 2011. Roles of cyclooxygenase 2 in sevoflurane- and olprinone-induced early phase of preconditioning and postconditioning against myocardial infarction in rat hearts. J. Cardiovasc. Pharmacol. Ther. 16, 72–78. Yue, L., Feng, J., Wang, Z., Nattel, S., 1999. Adrenergic control of the ultrarapid delayed rectifier current in canine atrial myocytes. J. Physiol. 516 (2), 385–398.