Role of mitochondrial dysfunction in cardiac glycoside toxicity

Abstract

Cardiac glycosides, which inhibit the plasma membrane Na(+) pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na(+) ([Na(+)](i)) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca(2+) ([Ca(2+)](m)) accumulation, and this impairment can be prevented by enhancing [Ca(2+)](m). Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca(2+) efflux, via inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na(+)](i) in a dose-dependent way, blunted [Ca(2+)](m) accumulation, decreased the NADH/NAD+redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca(2+) overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca(2+) retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca(2+)-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.

Figure 1

Representative recordings of [Na⁺]_i. Isolated cardiac myocytes were loaded with SBFI-AM. After 1 min recording, cells were treated with 100nM isoproterenol and ouabain at different concentrations as indicated for 5 min, and then were field-stimulated at 1 Hz for 3min (gray box). After stimulation, cells were recorded at rest for 1 min.

Figure 2

Effects of ouabain and CGP-37157 on mitochondrial Ca²⁺ accumulation, NADH production, and oxidative stress. In the presence of 100nM isoproterenol, control cells and ouabain-treated cells with or without CGP-37157 were stimulated at 1Hz for 3 min (grey box) followed by 2-min recovery at resting state. A. Representative recording of rhod-2 signals in control cell (left), cell treated with 1 µM ouabain (middle), and cell treated with ouabain plus CGP-37157 (right). B. Recording of NADH autofluorescence. C. Recording of DCF fluorescence.

Figure 3

Effects of ouabain and CGP-37157 on [Ca²⁺]_c cycling. Isolated cardiac myocytes were loaded with indo-1 AM. After 5 min treatment of 100nM isoproterenol with or without ouabain and CGP-37157, cells were field stimulated for 3 min. A) Representative [Ca²⁺]_c traces at the beginning (left panels) and end of stimulation (right panels). B) The average Δ[Ca²⁺]_c recorded at the midpoint of stimulation was is shown for control cells (n=3), ouabain treated cells (n=12), and ouabain plus CGP-37157 treated cells (n=12). * p<0.01 while compared to control; † p<0.05 while compared to ouabain treated group. C) The average of diastolic [Ca²⁺]_c recorded in control (isoproterenol only), ouabain-treated, and ouabain plus CGP-37157 -treated cells.

Figure 4

Effects of ouabain and CGP-37157 on DADs. In the presence of 100nM isoproterenol, current-clamped cells were treated with 1µM ouabain with or without 1µM CGP-37157 for 5 min, and action potentials (APs) were recorded at 1Hz for 3min. A) Representative AP traces. B) Amplitude of DADs. C) Probability of DAD-triggered AP activation. * p<0.01 compared to control; † p<0.01 compared to ouabain treated group.

Figure 5

Effects of ouabain and isoproterenol on cardiac function and oxygen consumption in isolated perfused hearts. A-C, Heart rates (A), ±dP/dt (B), and LVDP (C) measured at baseline and steady states after ouabain and isoproterenol application in groups with and without treatment of CGP-37157. * p<0.05 compared to baseline level; ‡ p<0.01 compared to that after ouabain application; † p<0.05 compared to that after isoproterenol application in the group without treatment of CGP-37157. D. Increases of LVDP by ouabain and isoproterenol. LVDPs measured at steady state after ouabain or isoproterenol application were compared to those before their application. * p<0.001. E. Measurements of oxygen consumption are displayed before and after application of ouabain and isoproterenol with or without CGP-37157, showing their effects on oxygen consumption.

Figure 6

Effects of ouabain and CGP-37157 on arrhythmia in isolated perfused heart. A-F. Representative tracings of LV pressure and ECG waveforms. A and B) Recording at the end of baseline recording; C and D) recording at the end of 10 min ouabain application; E and F) recording after isoproterenol administration. A, C, and E were recorded in a heart without CGP-37157 treatment. B, D, and F were recorded in a CGP-37157 treated heart. G. Comparison of arrhythmia scores. Arrhythmia scores were tabulated for the 10 min ouabain treatment period for hearts treated with (n=8) or without (n=8) CGP-37157. * p<0.01.

Figure 7

in vivo study of effect of CGP-37157 on ouabain-induced arrhythmia: A. representative traces of surface ECG recorded at different time points: baseline (upper), after ouabain administration (middle), and after isoproterenol administration (lower). B. heart rate at baseline, after ouabain administration, and after isoproterenol administration with (n=5) or without (n=5) pretreatment of CGP-37157. C. incidence of PVB after the administration of isoproterenol. In the group of iso, animals (n=4) were only administrated with 0.25mg/kg iso without pretreatment of ouabain.