Oxidoreductase regulation of Kv currents in rat ventricle

Abstract

Oxidative stress contributes to the arrhythmogenic substrate created by myocardial ischemia-reperfusion partly through a shift in cell redox state, a key modulator of protein function. The activity of many oxidation-sensitive proteins is controlled by oxidoreductase systems that regulate the redox state of cysteine thiol groups, but the impact of these systems on ion channel function is not well defined. Thus, we examined the roles of the thioredoxin and glutaredoxin systems in controlling K(+) channels in the ventricle. An oxidative shift in redox state was elicited in isolated rat ventricular myocytes by brief exposure to diamide, a thiol-specific, membrane-permeable oxidant. Voltage-clamp studies showed that diamide decreased peak outward K(+) current (I(peak)) evoked by depolarizing test pulses by 41% (+60 mV; p<0.05) while steady-state outward current (I(ss)) measured at the end of the test pulse was decreased by 45% (p<0.05). These electrophysiological effects were not prevented by protein kinase C blockers, but the tyrosine kinase inhibitors genistein or lavendustin A blocked the suppression of both K(+) currents by diamide. Moreover, inhibition of I(peak) and I(ss) by diamide was reversed by dichloroacetate and an insulin-mimetic. The effect of dichloroacetate to normalize I(peak) after diamide was blocked by the thioredoxin system inhibitors auranofin or 13-cis-retinoic acid, but I(ss) was not affected by either compound. A pan-specific inhibitor of glutaredoxin and thioredoxin systems, 1,3-bis-(2-chloroethyl)-1-nitrosourea, also blocked the dichloroacetate effect on I(peak) but only partially inhibited the recovery of I(ss). These data suggest that acute regulation of cardiac K(+) channels by oxidoreductase systems is mediated by redox-sensitive tyrosine kinase/phosphatase pathways. The pathways controlling I(peak) channels are targets of the thioredoxin system whereas those regulating I(ss) channels are likely controlled by the glutaredoxin system. Thus, cardiac oxidoreductase systems may be important regulators of ion channels affected by pathogenic oxidative stress.

Figure 1

Concentration-dependent inhibition of K⁺ currents by diamide. Ventricular myocytes were superfused with 10 or 100 µmol/l diamide and K⁺ currents recorded after washout. A: superimposed current traces recorded at test potentials from −40 to +60 mV. Mean I-V relations for I_peak (B) and I_ss (C) were measured from standard clamp protocols described in Methods. * = p<0.05 compared with control. Numbers in parentheses represent number of myocytes studied.

Figure 2

Inhibition of I_peak by H₂O₂. The same experimental protocol used for diamide was repeated with 1 or 10 µmol/l H₂O₂. Mean I-V relations are shown for I_peak (A) and I_ss (B). # = p<0.05 for both 1 and 10 µM H₂O₂ compared with control (panel A). * = p<0.05 compared with control (panel B).

Figure 3

Effects of kinase inhibitors on diamide-induced K⁺ current inhibition. Myocytes were treated with calphostin C (100 nmol/l), GF109203x (50 nmol/l), genistein (10 µmol/l) or lavendustin A (1 µmol/l) for 30 min before adding 100 µmol/l diamide for 20 min. I_peak (A) and I_ss (B) were recorded after washout of diamide. * = p<0.05 compared with control.

Figure 4

Recovery of I_peak (A) and I_ss (B) after diamide treatment by metabolic activators. Myocytes were exposed to 100 µmol/l diamide and then superfused with 1.5 mmol/l dichloroacetate (DCA) or 10 µmol/l bis-peroxovanadium- 1,10-phenanthroline (bpV(phen)). K⁺ currents were recorded after 15 min of exposure to metabolic activators. * = p<0.05 compared with control.

Figure 5

Block of DCA-mediated recovery of I_peak by the TrxR inhibitor auranofin (AF). Myocytes were pretreated with 10 nmol/l AF before superfusion with diamide followed by washout with DCA. AF blocked the effect of DCA to increase I_peak (A) but not I_ss (B). AF also blocked the effect of DCA to up-regulate I_peak after 10 µmol/l H₂O₂ (C). In panels A and C * = p<0.05 compared with diamide alone. In panel B * = p<0.05 compared with diamide-DCA.

Figure 6

Regulation of K⁺ currents by oxidoreductase systems. DCA-mediated up-regulation of I_peak (A) and I_ss (C) was assessed in myocytes pre-treated with 13-cis-retinoic acid (RA; 1 µmol/l) or 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU; 100µmol/l) for 30 min before diamide exposure. Up-regulation of I_peak by DCA was also assessed in myocytes exposed to 10 µmol/l H₂O₂ in place of diamide (B). * = p<0.05 compared with control. # = p<0.05 compared with diamide alone (panel C). D: TrxR and GR activities were assayed in control myocyte suspensions after treatment with inhibitors for 30 min. * = p<0.05 compared with no inhibition.