A key role for peroxynitrite‐mediated inhibition of cardiac ERG (Kv11.1) K ⁺ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations

Figures

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  Figure 1.. CO induces EAD-like arrhythmias in guinea pig myocytes. A) Example APs evoked in a guinea pig myocytes before (control) and after 3 min of exposure to the CO donor, CORM-2 (10 μM; left). Mean ±sem percentage changes in APD caused by 10 μM CORM-2 after 3 min (n = 14; right). B) Time course plot of the increase in APD50 caused by 10 μM CORM-2 (open symbols, n = 14) and 10 μM iCORM (solid symbols; n = 5). Each point is the mean ±sem percentage change in APD50 duration. C) Example APs evoked in a guinea pig myocyte before (control) and after 5 min of exposure to the iCORM (10 μM; left). Mean ±sem percentage changes in APD caused by 10 μM iCORM after 5 min (n = 5; right). D) Examples of evoked APs during more prolonged exposure to CORM-2. Note the emergence of EAD-like AP. E) Example APs evoked in a myocyte before (control) and after 4 min of exposure to E-4031 (1 μM; top). Mean ± sem percentage changes in APD caused by 1 μM E-4031 after 5 min (n = 6; bottom). F) Example APs evoked in a myocyte before (control) and after 3 min of exposure to CORM-2 (10 μM), then after 5 min exposure to NS1643 (1 µM) in the continued presence of CORM-2 (top). Mean ±sem percentage changes in APD caused…

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  Figure 2.. CO inhibits recombinant hERG. A) Illustration of the voltage-clamp protocol used to generate the fully activated current-voltage relationships in HEK293 cells (top). Example families of currents evoked by this protocol in HEK293 cells that stably express hERG before (control) and after (CORM-2) exposure of cells to 3 μM CORM-2 (bottom). B) Mean ± sem current voltage-relationships obtained from cells before (solid circles, n = 9) and during exposure to CORM-2 (3 μM; open circles, n = 9) or iCORM (3 μM; open triangles, n = 8). C) Concentration-response relationship obtained by determining the percentage inhibition of the hERG tail current caused by CORM-2 as determined by using a 2-step pulse protocol of 2 s of depolarization to +40 mV followed by a 2-s step to −40 mV from a holding potential of −80 mV and applied every 10 s. Each point plotted is the mean ± sem taken from between 3 and 6 cells. D) Time-series plot in which normalized peak tail current amplitudes, evoked by successive step depolarizations as indicated in panel C, are plotted against time (left). For the periods indicated by the horizontal bars, cell was exposed to 3 μM CORM-2 or 3 µM NS1643, as indicated.…

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  Figure 3.. CO inhibition of hERG involves mitochondrial ROS. A) Superimposed example currents from the same cell that illustrate the lack of effect CORM-2 (3 µM) after pretreatment of the cell with 100 nM ebselen for 30 min (left). Mean ± sem (n = 5) percentage inhibition of the peak tail current caused by 3 µM CORM-2 after pretreatment with 100 nM ebselen (right). B, C) Same as panel A, except that cells were pretreated with either antimycin A (B; n = 5) or myxothiazol (C; n = 6), both at 1 μM for 1 h at 37°C. N.s., not significant compared with paired t tests of amplitudes before and during CORM-2 application.

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  Figure 4.. CO inhibition of hERG involves the formation of peroxynitrite. A) Time-series plot in which normalized peak tail current amplitudes, evoked by step depolarizations from −80 to +40 mV followed by a repolarization to −40 mV, are plotted against time. For the period indicated by the horizontal bar, the cell was exposed to 3 μM CORM-2, as indicated. Before this recording, the cell was pretreated with 1 mM L-NAME for 1 h at 37°C. Superimposed example currents from the same cell that illustrate the lack of effect CORM-2 (3 µM) after pretreatment of the cell with L-NAME (inset). Mean ± sem (n = 7) percentage inhibition of the peak tail current caused by 3 µM CORM-2 after pretreatment with 1 mM L-NAME (right). B) Example recordings of APF fluorescence monitored in HEK293 cells that stably express hERG. At the point indicated by the arrow, cells were exposed to CORM-2, iCORM, and CORM-2 after pretreatment with 25 μM FeTPPS for 1 h at 37°C or CORM-2 after pretreatment with 1 mM L-NAME for 1 h at 37°C, as indicated. C) Bar graph plotting mean ± sem (determined from 5 or 6 recordings in each case) APF fluorescence determined at room temperature after exposure of cells to CORM-2, iCORM, or…

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  Figure 5.. hERG C723S mutant is insensitive to CO. Time-series plot in which normalized peak tail current amplitudes, evoked by successive step depolarizations from −80 to +40 mV followed by a repolarization to −40 mV, are plotted against time. For the period indicated by the horizontal bar, the cell was exposed to 3 μM CORM-2, as indicated. This recording was obtained from a HEK293 cell that was stably transfected with a C723S mutant form of hERG. Superimposed example currents from the same cell that illustrate the lack of effect CORM-2 (3 µM) on the C723S mutant form of the channel (inset). Mean ± sem (n = 5) percentage inhibition of the peak tail current caused by 3 µM CORM-2 in cells that express the C723S mutant form of hERG (right). N.s., not significant compared with paired t test of amplitudes before and during CORM-2 application.

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  Figure 6.. CO inhibits native ERG in guinea pig myocytes. A) Example currents in a guinea pig ventricular myocyte evoked by step depolarization from −40 to +20 mV, then repolarization to −40 mV. The current attributable to ERG channels is the transient outward current observed on repolarization. The horizontal line in all traces of this figure (A–C, E) indicate 0 current level. Two superimposed recordings are shown: before and during exposure of the myocyte to 1 µM E-4031. B, C). Same as panel A, except that cells were exposed either to CORM-2 (10 µM; B) or iCORM (10 µM; C). D) Bar graph plotting mean ± sem (n = 5–11 in each case) percentage inhibition of the peak tail current amplitude caused by application of E-4031, CORM-2, and iCORM (as exemplified in panels A–C). ***P < 0.001, paired Student’s t tests of control vs. drug treatments. E) Same as panel B, except the cell was pretreated for 1 h at 37°C with 25 µM FeTPPS (left). Mean ± sem (n = 9) percentage inhibition of the peak tail current caused by 10 µM CORM-2 in cells that were pretreated for 1 h at 37°C with 25 μM FeTPPS (right). F) Example APs evoked in a guinea pig myocyte previously pretreated for 1 h at 37°C with 25 μM FeTPPS…