Figure 5.

Na⁺ channel current (I_Na) and Na_v1.5 channel expression through modulation of the ubiquitin–proteasome pathway. (A) Representative I_Na families in the control condition, during the acute application of 20 µmol·L⁻¹ W-7, 0.06% SDS (sodium dodecyl sulphate, a proteasome activator) and 50 µmol·L⁻¹ MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide] in 4–5 min. (B) A summary of short-term (3–20 min) actions of W-7, SDS and MG132 on I_Na. I_Na at each condition was obtained at the test potential of −30 mV and normalized to the control value. (C) Representative I_Na families in vehicle (24 h), after the long-term action of 0.06% SDS (a proteasome activator), after the long-term action of SDS plus 10 µmol·L⁻¹ bepridil for 24 h, after the long-term action of 50 µmol·L⁻¹ MG132 for 24 h, and after the long-term action of 50 µmol·L⁻¹ MG132 plus 10 µmol·L⁻¹ bepridil (bep) or 20 µmol·L⁻¹ W-7 for 24 h. (D) I–V relationships constructed by using group data in vehicle, after the long-term action of 0.06% SDS for 24 h, after 0.06% SDS treatment for 24 h in the presence of 10 µmol·L⁻¹ bepridil, after long-term treatment of 50 µmol·L⁻¹ MG132 for 24 h, after bepridil (10 µmol·L⁻¹) treatment for 24 h in the presence of 50 µmol·L⁻¹ MG132 and after W-7 (20 µmol·L⁻¹) treatment for 24 h in the presence of 50 µmol·L⁻¹ MG132. (E) The maximum chord conductance in vehicle (24 h), after treatment with 0.06% SDS for 24 h, after treatment with 50 µmol·L⁻¹ MG132 for 24 h, after bepridil (10 µmol·L⁻¹) treatment for 24 h in the presence of 50 µmol·L⁻¹ MG132 and after W-7 (20 µmol·L⁻¹) treatment for 24 h in the presence of 50 µmol·L⁻¹ MG132. Numbers of experiments are shown in parentheses. (F) Expression of cardiac Na_v1.5 proteins assessed by Western blots analysis. Na⁺ channel proteins extracted from cardiomyocytes treated with vehicle, after treatment with 0.06% SDS for 24 h, after the treatment with 50 µmol·L⁻¹ MG132 for 24 h, after bepridil (10 µmol·L⁻¹) treatment for 24 h in the presence of 50 µmol·L⁻¹ MG132 and after W-7 (20 µmol·L⁻¹) treatment for 24 h in the presence of 50 µmol·L⁻¹ MG132. Representative protein expression levels of Na_v1.5 are shown in inset. (G) Effect of bepridil, W-7 and KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine] on 20S proteasome activity in vitro in comparison with that of MG132. 20S proteasome activities were assessed when cardiomyocytes were exposed to 10 µmol·L⁻¹ bepridil, 20 µmol·L⁻¹ W-7, 10 µmol·L⁻¹ KN93 and 50 µmol·L⁻¹ MG132 for 24 h, as relative to the values in the absence of drugs (1.0). **P < 0.01 compared with vehicle.