Yonago Acta medica 2013;56:93–95

Short Communication

Bepridil Suppresses Apoptosis in HL-1 Cardiac Atrial Myocytes Expressing
Mutant E334K cMyBPCRyo Endo,* Tomomi Notsu,† Mutsuo Mishima,† Kumi Morikawa,† Peili Li,† Nobuhito Ikeda,† Haruaki Ninomiya,‡
Yasuaki Shirayoshi† and Ichiro Hisatome†*Division of Anesthesiology and Critical Care Medicine, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan, †Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago 683-8503, Japan and ‡Department ofBiological Regulation, School of Health Science, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

MATERIALS AND METHODSABSTRACT
Besides its antiarrhythmic effect on atrial fibrillation,
bepridil protects tissue, yet its effect on apoptosis has
never been fully tested. We examine the effect of bepridil
on apoptosis of HL-1 cells expressing E334K myosinbinding protein C (MyBPC), a model cell of apoptosis.
Bepridil was compared with amiodarone, and its effects
on the expression of pro- and anti-apoptotic protein andapoptosis of HL-1 cells expressing mutant E334K MyBPC-green fluorescent protein (GFP) was analyzed using
Western blot and a flow cytometer. Bepridil decreased
the protein levels of both Bax and cytochrome c of cells
expressing E334K MyBPC-GFP with no changes in p53
and Bcl-2, while amiodarone decreased cytochrome cbut did not influence Bax except in its highest concentration. It also decreased the number of Annexin-V positivecells of HL-1 cells expressing E334K MyBPC-GFP, anddecreased apoptosis of HL-1 cells expressing E334K
MyBPC-GFP.

Cells culture and heterologous expression

HL-1 cardiac myocytes were provided by Dr. Claycomb(Louisiana State University) and cultured according toinstructions.5 cDNA encoding E334K cardiac MyBPC(cMyBPC) with a 6-myc tag at the N-terminus4 wasligated to pCS2+ at BamHI and XhoI sites to generate
plasmid expression vectors pCS-6myc-MYBPC3. To visualize the transfected cells, enhanced green fluorescentprotein (EGFP) cDNA was added to the carboxy terminus of E334K MyBPC cDNA. Transfection into HL-1
cells was performed using lipofectamin 2000 (Invitrogen,Carlsbad, CA) following the manufacturer’s instructions.
Western blotting

E334K MYBPC3 was transfected into HL-1 cells in theabsence or presence of either bepridil or amiodarone.
Protein extracts of cells were prepared 48 h post-transfection, as described elsewhere.5 Proteins were separated
by SDS-PAGE and electrotransferred to polyvinylidene
difluoride membrane. Membranes were probed with antibodies to actin (Calbiochem, La Jolla, CA), p53 (SantaCruz Biotechnology, Santa Cruz, CA), Bax (Santa Cruz),
cytochrome c (BD Biosciences, Franklin Lakes, NJ) or
Bcl-2 (Santa Cruz). They were developed using an ECL
system (Amersham Bioscience, Piscataway, NJ). The
intensities of the bands were quantified using National
Institute of Health image Software.

Key words: amiodarone; apoptosis; bepridil; HL-1 cell
Bepridil is classified as a Ca2+ channel blocker according to Vaughn-Williams’ classification.1 This agent also
has multi-ion channel blocking effects similar to thoseof amiodarone and is thought to be effective for atrial
fibrillation.2 Along with its electrophysiological actions
on atrial myocytes, bepridil showed beneficial effects intissue protection from ischemia.3 However, it remains
unknown whether bepridil protects the apoptosis ofatrial myocytes. Recently, we proposed cultured mouseatrial myocytes (HL-1 cells) expressing a Glu344Lys(E334K) missense mutation of myosin-binding protein
C (MyBPC) as the model to induce apoptosis.4 Thesecells exhibit apoptosis with increased pro-apoptotic proteins and decreased anti-apoptotic protein. In the present
study, we studied the effect of bepridil on apoptosis inHL-1 cells expressing mutant MyBPC in comparison
with amiodarone.

Annexin V staining and flow cytometry

Annexin V staining and flow cytometer of E334K
MYBPC3-GFP transfected cells were performed in theabsence and presence of either bepridil or amiodarone asreported elsewhere.4
Corresponding author: Ichiro Hisatome, MD
hisatome@med.tottori-u.ac.jp
Received 2013 October 9
Accepted 2013 November 1
Abbreviations: Af, atrial fibrillation; cMyBPC, cardiac MyBPC; EGFP, enhanced GFP; GFP, green fluorescent protein;
MyBPC, myosin-binding protein C93

R. Endo et al.

B

1.9%

* *Bepridil
29.6%

**

3.9%

3.3%

Annexin V-PE

Normalized ratio ofapoptosis

(%)

* * *

1.6%

MyBPC-GFP

MyBPC-GFP*(a)

Control
31.0%

* ***

Annexin V-PE

(b)

100

**

80

60

Control

BepridilFig. 1. Effects of bepridil and amiodarone on apoptosis in HL-1 cardiac atrial myocytes expressing E334K cMyBPC.
A: Effect of bepridil and amiodarone on the level of proteins regulating apoptosis in HL-1 cells expressing E334K cMyBPC in a dosedependent manner. Representative Western blot of pro-apoptotic proteins cytochrome c and Bax in HL-1 cells transfected with E334K
cMyBPC in the presence or absence of drugs as indicated. Beta-actin used as a control for protein loading. Summary of quantitative
densitometric scan of drugs on the level of Bax and cytochrome c; n = 8, *P < 0.05; **P < 0.01. cMyBPC, cardiac myosin-binding
protein C.
B: Effect of bepridil on the apoptotic cells expressing E334K cMyBPC-GFP.
(a) The representative immunofluorescence image of annexin V-positive cells in cells expressing E334K cMyBPC-GFP before and aftertreatment with bepridil.
(b) The percentage of annexin V-positive cells (n = 4) in those cells. Data are shown as mean (SEM), calculated by unpaired, two-tailed
Student’s t-test. *P < 0.05.
GFP, green fluorescent protein.

Used drugs

RESULTS AND DISCUSSION
Figure 1A shows the effects of bepridil or amiodarone
on the level of proteins that regulate apoptosis in HL-1
cells expressing E334K MyBPC. Bepridil decreased thelevels of Bax and cytochrome c, but not those of either
p53 or Bcl-2 in cells expressing E334K MyBPC (data notshown), which was also confirmed by quantitative analysis obtained from 6 different experiments. Amiodarone
also decreased the level of cytochrome c, while it decreased the level of Bax at the highest concentration.
Next, we examined the effect of bepridil on apoptosis ofHL-1 cells expressing E334K MyBPC-GFP as shown inFig. 1B. Bepridil reduced the number of annexin V-positive cells, which was confirmed by quantitative analysis(n = 4).
It has been reported that bepridil blocked the several
cardiac ion channels and was protective against atrial

Amiodarone and bepridil were used in this study. Bepridil was kindly provided by Daiichi Sankyo (Tokyo,Japan) and amiodarone was purchased from SigmaAldrich Japan (Tokyo).
Statistical analysis

Origin for Windows software version 7.0 (OriginLabCorporation, Northampton, MA) was used for statistical analysis. Differences between the 2 groups were assessed using the two-sample t-test. One-way analysis ofvariance with the Bonferroni test for post-hoc analysis
was used for multiple comparisons. All experimental
data were expressed as the mean (SEM). Differenceswith P values < 0.05 were considered significant.

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Effects of bepridil on apoptosis

fibrillation (Af). In addition to its acute action on ion
channels, bepridil reversed atrial electrical remodelingand L-type Ca2+ channel down regulation in a canine
model of persistent Af,6 suggesting that bepridil can exert beneficial effects on electrophysiological propertiesof atrial myocytes via modulating expression of ionicchannels. Some research has found that bepridil protects
cardiac myocytes from ischemia; impaired mitochondrial function, increasing reactive oxygen species, andapoptosis are involved in age-related atrial remodelingand Af susceptibility.7, 8 Other research has shown that it
reduces reactive oxygen species, and thus, is expected toreduce apoptosis. In the present study, bepridil reduced
the level of pro-apoptotic proteins (cytochrome c andBax) of cells expressing MyBPC-GFP, and this effect
was prominent in comparison with that of amiodarone.
The reduction of the level of cytochrome c suggested
protective effect of bepridil that was mediated throughthe mitochondrial pathway. Although bepridil inhibited
Na+/Ca2+ exchanger and suppressed apoptosis of neurons,9 this is the first report demonstrating the effectof bepridil on apoptosis in a cellular model expressing
E334K MyBPC.

2 Nakazato Y, Yasuda M, Sasaki A, Iida Y, Kawano Y,
Nakazato K, et al. Conversion and maintenance of sinus
rhythm by bepridil in patients with persistent atrial fibrillation.
Circ J. 2005;69:44-8. DOI: 10.1253/circj.69.44.
3 Massingham R, Van Zwieten PA. Bepridil: a pharmacological reappraisal of its potential beneficial effects in angina andtissue protection following ischemia. Cardiovasc Drugs Ther.
1989;3:731-42. DOI: 10.1007/BF01857623.
4 Bahrudin U, Morisaki H, Morisaki T, Ninomiya H, Higaki
K, Nanba E, et al. Ubiquitin-proteasome system impairment
caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic
cardiomyopathy. J Mol Biol. 2008;384:896-907. DOI: 10.1016/j.jmb.2008.09.070.
5 White SM, Constantin PE, Claycomb WC. Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes
for studies of cardiac muscle cell structure and function. AmJ Physiol Heart Circ Physiol. 2004;286:H823-9. DOI: 10.1152/ajpheart.00986.2003.
6 Nishida K, Fujiki A, Sakamoto T, Iwamoto J, Mizumaki K,
Hashimoto N, et al. Bepridil reverses atrial electrical remodeling and L-type calcium channel downregulation in a canine
model of persistent atrial tachycardia. J Cardiovasc Electrophysiol. 2007;18:765-72. DOI: 10.1111/j.1540-8167.2007.00833.
x.
7 Mihm MJ, Yu F, Carnes CA, Reiser PJ, McCarthy PM, Van
Wagoner DR, et al. Impaired myofibrillar energetics andoxidative injury during human atrial fibrillation. Circulation.
2001;104:174-80. DOI: 10.1161/ 01.CIR.104.2.174.
8 Aimé-Sempé C, Folliguet T, Rücker-Martin C, Krajewska M,Krajewska S, Heimburger M, et al. Myocardial cell death infibrillating and dilated human right atria. J Am Coll Cardiol.
1999;34:1577-86. DOI: 10.1016/S0735-1097(99)00382-4.
9 Momeni HR, Jarahzadeh M. Effects of a voltage sensitive calcium channel blocker and a sodium-calcium exchanger inhibitor on apoptosis of motor neurons in adult spinal cord slices.
Cell J. 2012;14:171-6. Epub 2012 Dec 12. PMID: 23508879.

The authors declare no conflict of interest.
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