Bepridil up‐regulates cardiac Na+channels as a long‐term effect by blunting proteasome signals through inhibition of calmodulin activity

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Abstract
Background and purpose: 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 Nav1.5 channel.Experimental approach: 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.Key results: Treatment of cardiomyocytes with 10 µmol·L−1bepridil for 24 h augmented Na+channel current (INa) 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, theINaincrease due to bepridil was larger than the increase without cycloheximide. Bepridil and W‐7 significantly slowed the time course of Nav1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Nav1.5 were not modified. Bepridil and W‐7 did not increaseINaany 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 and implications: We conclude that long‐term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Nav1.5 α‐subunit, which in turn increased Na+current.

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