Inhibition of the voltage-gated potassium channel Kv1.5 by hydrogen sulfide attenuates remodeling through S-nitrosylation-mediated signaling

Figures

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  Fig. 1. Electrical and cellular remodeling in HL-1 cardiomyocytes via regular interval pacing AP recordings from control and paced HL-1 cardiomyocytes. a Representative spontaneous AP recordings from a control unstimulated cell (upper) and a cell that had been subjected to high frequency interval pacing (lower). b Superimposed spontaneous APs (total 36) from a typical control (upper) or paced (lower) HL-1 myocyte, demonstrating that prior interval pacing results in shortening of spontaneous APs and a decrease in spontaneous AP frequency. c Example showing irregularity of intervals between spontaneous APs apparent in some cells after interval pacing. d The probability density histograms for these intervals (N = 17, 8.6 ± 1.4 sec). e Representative western blots for native IKur/Kv1.5 after various interval pacing time periods (0–8 h) showing increased protein level when compared to control unstimulated conditions. f Summary mean (±s.e.m) band intensities as determined using ImageJ software (n = 6–7 experiments, one-way ANOVA with post hoc Bonferroni correction). g Representative western blots for H2S producing enzymes at different pacing time periods (0–8 h) showing increased levels of…

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  Fig. 2. H2S inhibits recombinant Kv1.5. a, b Examples of time-series plots illustrating normalized peak current amplitudes evoked by step depolarizations from −70mV to +50 mV in HEK293 cell expressing hKv1.5 before, during (horizontal bar) and after application of the fast-releasing H2S donor NaHS. or the slow-releasing H2S donor GYY4137 via perfusate. Insets show example currents under the conditions described; scale bars: 2 nA (vertical) and 50 ms (horizontal). c, d Mean ( ± s.e.m., values from 4–7 cells), concentration-response relationship illustrating the effects of H2S donor NaHS or GYY4137 on hKv1.5 expressed in HEK 293 cells. Inhibition was determined using currents evoked by step-depolarizations from −70mV to +50 mV. Curves obtained by fitting data to Boltzmann function. e Mean ( ± s.e.m.; n = 9; *P < 0.05) current-density versus voltage relationships before (open circles) and during (solid circles) exposure to NaHS (300 µM). f Families of currents evoked in a HEK293 cell stably expressing hKv1.5 before (upper) and during exposure to NaHS (300 µM; lower). Currents evoked by step-depolarizations, applied up to +80 mV in 10 mV increments from a holding potential of −70 mV. Scale…

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  Fig. 3. H2S inhibits native Kv1.5/ IKur currents in HL-1 and resulted in prolongation of AP. a Current-density versus voltage relationships before (open circles) and during (solid circles) exposure to H2S-releasing donor NaHS (300 µM; n = 8, mean ± s.e.m.). b Families of outward K+ currents recorded in an example HL-1 cell, before (right) and during exposure to H2S (left, 300 µM). c Bar graph showing percentage normalized mean (±s.e.m.) effect of NaHS (300 µM) and Kv1.5 inhibitor DPO-1 (1 µM) on outward K+ currents peak amplitude measured at +50 mV. d Examples showing control recordings (black) and the effect of H2S (grey, left) or DPO-1 (grey, right). Scale bars apply to all families of currents 0.1 nA (vertical) and 50 ms (horizontal). e Cumulative APD90 data (mean, ± s.e.m,) recorded from HL-1 cells or in adult rat atrial cells before (control, unfilled) and during application of 300 μM NaHS (filled). f Superimposed evoked APs (N = 20) before (black) and during 100 µM NaHS (grey) perfusion in HL-1 cells or (g) in rat atrial myocyte. For all panels paired t test; *P < 0.05, **P < 0.01.

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  Fig. 4. H2S reduced electrical and cellular remodeling in paced HL-1 atrial cardiomyocytes. a Representative AP recordings in spontaneously beating HL-1 cells from control cells that were not paced during the 8 h culture period (upper), cells that were paced at 5 Hz for 30 min regular intervals (middle) and paced cells that were incubated with slow-releasing H2S donor GYY4137 (lower). b Cumulative data showing mean (±s.e.m.) APD under control conditions (n = 11), in paced cells (n = 10), and in paced cells incubated with GYY4137 (n = 11).*P < 0.05, **P < 0.01. c Representative western blots for native IKur/Kv1.5, and H2S producing enzymes CBS, CTH and MPST at different pacing time periods (2–8 hr) in the absence of GYY4137 or with incubation with GYY4137 (horizontal line), compared to control unstimulated conditions (0 h). β-actin was used as loading control in all western blots. d Mean (±s.e.m) percentage change of band intensities following (n = 4 experiments) at different pacing time periods (2–8 hr) in the absence of GY4137 or with GYY4137 incubation, as indicated by the horizontal line.

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  Fig. 5. Inhibition of Kv1.5 by H2S requires NO formation and nitrosylation of hKv1.5. a Cumulative data showing the percentage inhibition (mean ± s.e.m.) induced by the H2S donor NaHS on normalized hKv1.5 peak amplitude in HEK293, currents evoked by repeated step depolarizations from −70 mV to +50 mV (100 ms duration, 0.2 Hz) in untreated (n = 8), L-NAME (1 mM; 1 h, 37 °C; n = 8) or triciribine pre-treated cells (5 µM; 1 h, 37 °C; n = 8) **P < 0.01, ***P < 0.001. b, c Example time-series plots showing the lack of effect of H2S donor NaHS during perfusion on normalized peak current amplitudes obtained from either L-NAME or Triciribine pre-treated HEK293 cell stably expressing human Kv1.5 (hKv1.5). Inset shows example currents before and during NaHS exposure (horizontal scale bar = 50 ms). d Representative western blots showing the time dependent effect of NaHS treatment on eNOS phosphorylation at Ser 1177 (upper), eNOS expression (middle). β-actin used as loading control (lower). Cell lysates obtained from H2S treated HEK293 cell stably expressing hKv1.5 at the time indicated, control represent cells not exposed to H2S. e Western blot showing detection of Kv1.5 nitrosylation via the…