Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity.
OBJECTIVE
Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na+ channel. However, long-term effects of bepridil on the Na+ channel remain unclear. We explored the long-term effect of bepridil on the Na+ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na(v)1.5 channel.


METHODS
Na+ currents were recorded by whole-cell voltage-clamp technique. Na+ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis.


RESULTS
Treatment of cardiomyocytes with 10 micromol.L(-1) bepridil for 24 h augmented Na+ channel current (I(Na)) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca2+/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I(Na) increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na(v)1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na(v)1.5 were not modified. Bepridil and W-7 did not increase I(Na) any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner.


CONCLUSIONS
We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na(v)1.5 alpha-subunit, which in turn increased Na+ current.
Department of Pathophysiology, Oita University School of Medicine, Oita 879-5593, Japan.
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L
Kang
M Q
MQ
Zheng
M
M
Morishima
Y
Y
Wang
T
T
Kaku
K
K
Ono
D000818:Animals; D000831:Animals, Newborn; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology*; D015764:Bepridil / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000494:pharmacology; D006801:Humans; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D061988:Proteasome Inhibitors; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015222:Sodium Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D015854:Up-Regulation
D000889:Anti-Arrhythmia Agents; D002147:Calmodulin; D020536:Enzyme Activators; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D061988:Proteasome Inhibitors; D021122:Protein Subunits; C568320:SCN5A protein, human; C568322:Scn5a protein, rat; D015222:Sodium Channels; D015764:Bepridil; D046988:Proteasome Endopeptidase Complex