Effects of MWCNTs on the <i>I</i>_to of isolated rat ventricular myocytes.

<p><b><i>A</i></b>, a typical example of <i>I</i>_to recorded from ventricular myocytes. <b><i>B</i></b>, voltage-dependent activation and inactivation curves of <i>I</i>_to from rat ventricular myocytes with or without (control) MWCNTs (20 µg/ml) incubation. <b><i>C</i></b>, comparison of the <i>I</i>_to traces and τ_decay in rat ventricular myocytes with (<i>thick</i>) or without (<i>thin</i>) MWCNTs treatment. Ventricular myocytes were depolarized from −50 mV to +50 mV. <b><i>D</i></b>, statistical data of the τ_decay of ventricular myocytes with or without MWCNTs treatment. <b><i>E</i></b>, <i>I</i>_to recovery curves. <b><i>F</i></b>, statistical τ_recovery bar graphs of control cells and MWCNT-treated cells. * <i>P</i><0.05 <i>vs.</i> control.</p

The action potential-shaping and arrhythmogenic effects of MWCNTs.

<p><b><i>A</i></b>, an overlap of the action potentials of isolated ventricular myocytes at baseline and after MWCNT treatment. <b><i>B</i></b><i> and </i><b><i>C</i></b>, statistical data showing the effects of MWCNTs on the APD and APA, respectively. <b><i>D</i></b>, typical surface ECGs and ventricular MAPs recorded from an in-situ heart showing that intravenous MWCNTs soon induced bradycardia, AV block and cardiac asystole. P, QRS and T represented the P wave, QRS complex and T wave, respectively.</p

Effects of MWCNTs on Kv4.2 trafficking in HEK293 cells.

<p><b><i>A</i></b>, an original recording of flow cytometry showing the fluorescence of Flag which represented the population of Kv4.2 in cell membrane. A leftward shift of the trace meant a reduction of fluorescence intensity in membrane and vise versa. Untransfected HEK293 cells served as negative control. <b><i>B</i></b>, ratio of mean fluorescence intensity of Flag to GFP in HEK293 cells expressing Kv4.2 with or without KChIP2 upon MWCNTs treatment from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101545#pone-0101545-g007" target="_blank">Figure 7A</a>. <b><i>C</i></b>, aliquots of cell lysate from HEK293 cells expressing Kv4.2 and KChIP2 were mixed with either anti-Kv4.2 antibody or anti-KChIP2 antibody and precipitated with protein A-sepharose. Immune complexes were resolved by SDS-PAGE. The immunoprecipitating antibodies were indicated above each lane, and detecting antibodies were shown at the left side. <b><i>D</i></b>, effects of MWCNTs treatment on the ratio of KChIP2 to Kv4.2 with anti-Kv4.2 antibody as bait for co-IP in HEK293 cells and cell lysate. MWCNTs were applied to the culture medium and cell lysate for 6 h, respectively. <b><i>E</i></b>, statistical results of the effects of MWCNTs on the ratio of KChIP2 to Kv4.2 in HEK293 cells expressing Kv4.2 and KChIP2. * <i>P</i><0.05, ** <i>P</i><0.01 <i>vs.</i> control. <b><i>F</i></b>, confocal images showing the membranous and intracellular distributions of Kv4.2. GFP (<i>green</i>) and anti-Flag <i>(red</i>) represented the total and membranous Kv4.2, respectively. Note that MWCNT-treated cells showed reduced membranous Kv4.2 compared with control cells. Scale bars  =  50 µm.</p

The expression of Kv4.2 and KChIP2 in HEK293 cells.

<p><b><i>A</i></b>, diagram for Flag-Kv4.2-GFP oriented in the plasma membrane. Flag and GFP were tagged in the extracellular S1-S2 loop and the C-terminal, respectively. <b><i>B</i></b>, Western blotting analysis of HEK293 cells expressing Kv4.2 with or without the expression of KChIP2. The plasmids transfected in HEK293 cells were indicated above each lane. The detecting antibodies were indicated at the right side. Lane 1 showed the lysate from cells expressing the corresponding vector as control. Lane 2 and 3 represented cells expressing Kv4.2 alone or with KChIP2, respectively. <b><i>C</i></b>, an example of <i>I</i>_Kv4.2 recorded in HEK293 cells expressing Flag-Kv4.2-GFP. No significant difference of <i>I</i>_Kv4.2 was found between Flag-Kv4.2-GFP-expressing cells and cells expressing untagged Kv4.2 (not shown). <b><i>D</i></b>, confocal images of HEK293 cells transfected with Flag-Kv4.2-GFP, showing subcellular localization of Kv4.2, with (P (+)) or without (P (−)) membrane permeabilization. The surface population of Kv4.2 was visualized by anti-Flag antibody (<i>red</i>) and the whole Kv4.2 was indicated by GFP (<i>green</i>). The nuclei were stained by diamidino-phenyl-indole (DAPI) (<i>blue</i>). Note that anti-Flag antibody detected only the membranous Kv4.2 when without permeabilization, while it detected the overall Kv4.2 after permeabilization. The merged images showed perfect co-localization of Flag-Kv4.2-GFP detected by anti-Flag antibody and GFP, respectively. Scale bars  =  50 µm.</p

Effects of differently modified MWCNTs on the recovery kinetics of Kv4.2.

<p>MWCNTs were added to the pipette solution. <b><i>A</i></b> and <b><i>B</i></b>, examples of recovery currents recorded from HEK293 cells expressing Kv4.2 with or without treatment of MWCNTs. <b><i>C</i></b>, normalized <i>I</i>_Kv4.2 in HEK293 cells upon different stimuli. <b><i>D</i></b>, statistical data based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101545#pone-0101545-g003" target="_blank">Figure 3C</a>. * <i>P</i><0.05, ** <i>P</i><0.01 <i>vs.</i> Kv4.2.</p

Effects of MWCNTs on Kv4.2 and KChIP2 protein expression in transfected HEK293 cells.

<p><b><i>A</i></b>, Western blotting analysis showing changes of Kv4.2 and KChIP2 in two cell lines. <b><i>B</i></b>, statistical histograms from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101545#pone-0101545-g006" target="_blank">Figure 6A</a> showing the fold changes after 6 h-treatment with MWCNTs. <b><i>C</i></b>, biotinylation results showing the surface population of Kv4.2, with the expression of endogenous Na⁺/K⁺-ATPase (NKP) as loading control. <b><i>D</i></b>, quantification histograms indicating the fold changes of the surface population of Kv4.2. * <i>P</i><0.05 <i>vs</i>. control.</p

Flag and GFP tags did not affect the kinetics of Kv4.2 channel in HEK293 cells.

<p>The channel kinetics parameters of HEK293 cells expressing wild-type Kv4.2 or Flag-Kv4.2-GFP were examined in a whole-cell configuration. V_1/2-act, membrane potentials at half maximum activation. V_1/2-inact, membrane potentials at half maximum inactivation. τ_decay, the decay time constant of inactivation. τ_recovery, the recovery time constant from inactivation. These parameters between the two cell lines did not yield statistical significance.</p

Effects of longer (6 h) exposure to MWCNTs on Kv4.2 channel kinetics in HEK293 cells.

<p><b><i>A</i></b>, I-V curves of <i>I</i>_Kv4.2 at different conditions. <b><i>B</i></b>, the voltage-dependent activation and inactivation curves fitted with Boltzmann equation. <b><i>C</i></b> and <b><i>D</i></b>, effects of MWCNTs on the decay kinetics of HEK293 cells expressing Kv4.2 with or without KChIP2. ** <i>P</i><0.01 <i>vs.</i> control. <b><i>E</i></b> and <b><i>F</i></b>, the recovery curve and the statistical recovery time constant (τ_recovery), respectively. ^**<i>P</i><0.01 <i>vs.</i> control.</p

Effects of MWCNTs on the activation and inactivation kinetics of Kv4.2.

<p><b><i>A</i></b> and <b><i>B</i></b>, examples of activation (<i>A</i>) and inactivation (<i>B</i>) currents from transfected HEK293 cells. <b><i>C</i></b> and <b><i>D</i></b>, Boltzmann equation-fitted activation and inactivation curves, respectively. <b><i>E</i></b>, <i>I</i>_Kv4.2 obtained by depolarizing the cell from −90 mV to +50 mV, showing accelerated decay upon MWCNTs treatment in HEK293 cells co-expressing Kv4.2 with KChIP2. <b><i>F</i></b>, statistical data of decay time constants (τ_decay) in various conditions. Abbreviations: C, carboxylated MWCNTs; P, pristine MWCNTs; A, aminated MWCNTs. ^**<i>P</i><0.01 <i>vs.</i> Kv4.2. ^##<i>P</i><0.01 <i>vs.</i> KChIP2.</p

Zacopride did not affect hypoxia-induced and glibenclamide-sensitive currents in isolated rat ventricular myocytes.

<p>(A) The I-V curves of the measured currents. a, baseline. b, hypoxia. c, 10 μmol/L glibenclamide. (B) Time course of the membrane currents measured at 20 mV under hypoxic condition. One μmol/L zacopride and 10 μmol/L glibenclamide were applied in succession.</p