Carbon monoxide induces cardiac arrhythmia via induction of the late Na+ current

Abstract

Rationale: Clinical reports describe life-threatening cardiac arrhythmias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown.

Objectives: To understand the cellular basis of CO-induced arrhythmias and to identify an effective therapeutic approach.

Methods: Patch-clamp electrophysiology and confocal Ca(2+) and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo.

Measurements and main results: CO increased the sustained (late) component of the inward Na(+) current, resulting in prolongation of the action potential and the associated intracellular Ca(2+) transient. In more than 50% of myocytes these changes progressed to early after-depolarization-like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na(+) channel, Na(v)1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor l-NAME, and reversed by ranolazine, an inhibitor of the late Na(+) current. Ranolazine also corrected QT variability and arrhythmias induced by CO in vivo, as monitored by telemetry.

Conclusions: Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitrosylation of Na(V)1.5 and to induction of the late Na(+) current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.

Figure 1.

Carbon monoxide (CO) disrupts intracellular Ca²⁺ handling in ventricular myocytes. Line-scan images of [Ca²⁺]_i in a fluo-3 loaded myocyte before (control) and during superfusion with CO (87.6 ± 4.2 μΜ) (A) or CO-releasing molecule (CORM)-3 (20 μM) (B) for 2 and 6 minutes. Arrowhead indicates time of electrical stimulation. Scale bars apply to all images. Bottom, normalized superimposed line-scan images illustrating the effect of CO (A) or CORM-3 (B) on the descending phase of the Ca²⁺ transient. In approximately 50% of cells the increase in duration of the descending phase developed into a plateau with clear [Ca²⁺]_i oscillations. Bar graph, mean (± SEM bars; n = 14) increase of Ca²⁺ transient duration (at 50% of maximum) caused by CO. **P < 0.01, ***P < 0.001, paired t test (A). (B) As A, except the myocyte was exposed to 20 μM CORM-3 rather than CO. Bar graph shows mean (± SEM bars; n = 14) increase of Ca²⁺ transient duration (at 50% of maximum) caused by CORM-3. **P < 0.01, ***P < 0.001, paired t test.

Figure 2.

Carbon monoxide (CO) prolongs the cardiac action potential (AP) by increasing the amplitude of the late Na⁺ current. (A) APs recorded before (black) and during (red) perfusion with CO-releasing molecule (CORM)-2 (30 μM). Note reduced peak amplitude and increased duration (upper traces) or oscillations (lower traces). Bar graph, mean % change (± SEM; n = 6) in AP duration at the points indicated. **P < 0.01 (paired t test). (B) Na⁺ currents evoked by depolarizations from −80 to −30 mV before and during exposure to 30 μM CORM-2, CO (87.6 ± 4.2 μM), or 30-μM inactive breakdown product (RuCl₂[DMSO]₄) of CORM-2 (iCORM), as indicated. Scale bars apply to all traces. Note enhanced late Na⁺ current caused by CO, shown more clearly in traces below. Bar graph, mean (± SEM; n = 8 for CORM-2, 7 for CO, and 6 for iCORM) % change in peak and late Na⁺ current evoked by CORM-2, CO, and iCORM, as indicated. **P < 0.01, paired t test.

Figure 3.

Inhibiting nitric oxide (NO) formation prevents the proarrhythmic effects of carbon monoxide (CO). (A) Top, line scan images of [Ca²⁺]_i before and during exposure to CO (87.6 ± 4.2 μM). In this experiment, 1 mM l-NAME was present throughout and for 120 minutes before recording. Bottom, normalized fluorescence plotted as a function of time. Bar graph, mean (± SEM; n = 6) duration of Ca²⁺ transients (at 50% of maximum) caused by CO in the presence of l-NAME. (B) Na⁺ currents recorded before and during exposure to CO-releasing molecule (CORM)-2 (30 μM), as indicated. Throughout the experiment, and 30 minutes before recording, the myocyte was exposed to 1 mM l-NAME. Bar graph, mean (± SEM; n = 7) % change in peak and late Na⁺ current amplitudes evoked by 30 μM CORM-2 in the absence and presence of l-NAME, as indicated. **P < 0.01, paired t test. (C) Action potentials (APs) recorded before and during perfusion with 30 μM CORM-2. l-NAME (1 mM) was present throughout and 30 minutes before recording. Bar graph, mean % change (± SEM; n = 7) in AP duration at points indicated.

Figure 4.

Carbon monoxide (CO) induces nitric oxide (NO) formation and S-nitrosylation of Nav1.5. (A) Top, fluorescence images of myocytes loaded with the NO indicator, DAF-2: left, superfusion with CO (87.6 ± 4.2 μM) alone; right, superfusion with CO after preexposure to 1 mM l-NAME. Bottom, graph plotting mean (± SEM; n = 6) changes in DAF-2 fluorescence during exposure to CO in the absence or presence l-NAME, as indicated. *P < 0.05; **P < 0.01, unpaired t tests. (B) Identification of S-nitrosylated Na_v1.5 in cardiac myocyte extracts made from untreated myocytes or myocytes after treatment with inactive breakdown product (RuCl₂[DMSO]₄) of CORM-2 (iCORM) or CORM-2 (each at 30 μM), or CysNO (50 μM). (C) Effects of the reactive disulfide 2,2'-dithiobis(5-nitropyridine) (5-NO-2-PDS); (50 μM) on the peak and late Na⁺ current in rat ventricular myocytes: example currents evoked by step depolarizations from −80 to −30 mV are shown before (control) and during exposure to 5-NO-2-PDS. Bar graph shows mean (± SEM; n = 5 cells) percent change in peak and late current amplitude caused by 5-NO-2-PDS. (D) Line profiles obtained from line-scan images illustrating the effect of CO on cardiac myocytes. Top, evoked Ca²⁺ transients before and during exposure to CO (87 μM), applied for the period indicated by the bar above the trace. Two evoked transients are shown on an expanded time scale to highlight the early after-depolarizations (EAD)–like changes in transients typical of the effects of CO. Bottom, experiment conducted exactly as shown in the upper example, except that 2 mM dithiothreitol (DTT) was present throughout the experiment. Note the lack of appearance of EAD-like events.

Figure 5.

Ranolazine (Ran) reverses the cellular effects of carbon monoxide (CO). (A) Line scan images of [Ca²⁺]_i before (control) and during superfusion with CO (87.6 ± 4.2 μΜ +CO) then during addition of 20 μM ranolazine (+CO +Ran). Right, normalized fluorescence taken from the same three images. Note CO induced oscillations, which were abolished by Ran. Below, bar graph, mean (± SEM; bars, n = 10) increase in transient duration (at 50% of maximum) caused by CO, and reversal by Ran ( 20 μM). **P < 0.01, paired t test (B) Na⁺ currents evoked by depolarizations from −80 to −30 mV before and during exposure to 30 μM CO-releasing molecule (CORM)-2 alone, then in the additional presence of Ran. Bar graph; mean (± SEM; n = 7) % change in peak and late Na⁺ current evoked by CORM-2, before and during exposure to Ran. **P < 0.01. (C) Action potentials (APs) before and during perfusion with 30 μM CORM-2 alone or in the presence of Ran (20 μM). Bar graph plots mean % change (± SEM; bars) in normalized AP duration (n = 7) at the points of the AP indicated. *P < 0.05; **P < 0.01 (paired t test).

Figure 6.

Carbon monoxide (CO) increases the QT interval and variability in vivo. (A) Typical examples of ECGs recorded in sham, CO-exposed, and CO-exposed ranolazine (Ran)-treated rats. (B) Bar graph showing mean (± SEM; bars) QTc intervals under control conditions, or after CO exposure (30 and 60 min) and after isoproterenol challenge (1 mg⋅kg⁻¹ intraperitoneally), as indicated, in sham (black bars), CO-exposed (white bars), and CO-exposed Ran-treated groups (hatched bars). **P < 0.01 and ***P < 0.001 versus sham; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus preceding period in the same group; ^ΨΨP < 0.01 versus untreated Ran animals. (C) Representative traces of QT variability, presented as Poincaré plots.

Figure 7.

Isoproterenol potentiates the proarrhythmic effects of carbon monoxide (CO). (A) Mean (± SEM; bars) number of ventricular ectopic beats (VEB) occurring spontaneously or during isoproterenol challenge in the same experimental groups as indicated in Figure 6B. (B). Percentage of animals developing ventricular tachycardia (VT), ventricular fibrillation (VF), and sudden arrhythmic death during the test of arrhythmogenic susceptibility by isoproterenol injection (1 mg/kg intraperitoneally) in the same experimental groups as indicated in Figure 6B. ***P < 0.001 versus sham; ^##P < 0.01 and ^###P < 0.001 versus preceding period in the same group; ^ΨP < 0.05, ^ΨΨP < 0.01, and ^ΨΨΨP < 0.001 versus untreated ranolazine animals.