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Role of mitochondrial dysfunction in cardiac glycoside toxicity

https://doi.org/10.1016/j.yjmcc.2010.06.012Get rights and content

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 Ca2+ ([Ca2+]m) accumulation, and this impairment can be prevented by enhancing [Ca2+]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 Ca2+ efflux, via inhibition of the mitochondrial Na+/Ca2+ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na+]i in a dose-dependent way, blunted [Ca2+]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 Ca2+ 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 Ca2+ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca2+-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.

Research highlights

Background: ► Cardiac glycosides have been used for centuries to treat heart failure, yet their low therapeutic index and potential arrhythmogenic actions have limited their therapeutic potential. ► Glycosides work primarily by inhibiting the plasma membrane Na+ pump, which can help increase sarcoplasmic reticular Ca2+ loading. However, high cytosolic Na+ also inhibits mitochondrial Ca2+ signaling, which can lead to a mismatch of energy supply and demand.

Results and significance: ► The present study shows that the cardiac glycoside ouabain increases cytosolic Na+, blunts mitochondrial Ca2+ accumulation during increased work (β-adrenergic stimulation and electrical pacing), causes net oxidation of the NAD(P)H pool, increases reactive oxygen species accumulation, and causes triggered arrhythmias. ► The mitochondrial dysfunction caused by ouabain could be prevented by enhancing mitochondrial Ca2+ uptake with an inhibitor (CGP37157) of the mitochondrial Na+/Ca2+ exchanger (mNCE). ► CGP37157 effectively preserved mitochondrial function during ouabain administration, potentiating the positive inotropic actions of the glycoside (and β-adrenergic agonists) and mitigating the toxic effects in cells, perfused hearts, or intact animals. ► The results suggest that partial mNCE inhibition may represent a novel strategy for improving cardiac glycoside therapy in the context of heart failure.

Introduction

Cardiac glycosides have been widely used in the treatment of heart failure (HF) for more than 200 years, and they are one of the 4 categories of drug that are recommended for routine use to treat HF by The American College of Cardiology/American Heart Association Joint Guidelines[1]. Treatment with digitalis glycosides can improve HF symptoms, increase cardiac output, enhance quality of life, and decrease clinical decompensation and hospitalization; however, the digitalis toxicity-related death in HF patients, especially in the subgroup with high serum digoxin concentration, undermines the beneficial effect of digitalis treatment on total mortality rates (reviewed in [2]).

The primary action of cardiac glycosides is their ability to inhibit Na+/K+-ATPase (NKA), which elicits multiple effects on cardiac physiology and pathology (reviewed in [3]). Inhibition of NKA on the sarcolemma of cardiac myocytes has a positive inotropic effect, mediated by an elevation of intracellular Na+ ([Na+]i). Elevated [Na+]i increases sarcoplasmic reticulum (SR) Ca2+ load by affecting the activity of the sodium/calcium exchanger (NCX) on the sarcolemma, as a consequence of a reduction of the driving force for Ca2+ extrusion and/or an increase in Ca2+ influx via the NCX. The resulting increase of SR Ca2+ load is responsible not only for the inotropic effect but also for the arrhythmogenic effects of glycosides, the major adverse effect of digitalis drugs. Besides its inotropic effect, clinical trials have shown that digitalis glycosides could also reduce plasma norepinephrine levels [4], [5], [6], serum aldosterone [4], [6], [7], and plasma renin activity [4], [7] in patients with HF. The beneficial effects of glycosides on HF are probably attributable to both their inotropic and neurohormonal effects. The adverse effects of glycoside have also been well documented, which include cardiac arrhythmias, gastrointestinal symptoms, and central nervous system abnormalities [1].

Our recent studies have led us to speculate that glycosides might impair mitochondrial energetics in cardiac myocytes due to elevated [Na+]i[8], [9]. Mitochondrial Ca2+ ([Ca2+]m) homeostasis plays a central role in energy supply and demand matching. Increased cardiac work leads to an increase in [Ca2+]m accumulation[8], which is critical for maintaining NADH/NAD+ redox potential [8], [9] by activating several enzymes in the tricarboxylic acid cycle [10]. Elevated [Na+]i blunts [Ca2+]m accumulation by activating the mitochondrial Na+/Ca2+ exchanger (mNCE), the major [Ca2+]m efflux pathway, and therefore it mediates net oxidation of NADH during increased work. Our recent studies have demonstrated this adverse effect of elevated [Na+]i on mitochondrial energetics in normal cardiac myocytes, with [Na+]i elevated artificially using the patch clamp technique, and in myocytes isolated from failing hearts, with a chronic pathological elevation of [Na+]i[8], [9]. Our previous studies also showed the potential therapeutic effect of CGP-37157, an inhibitor of mNCE. CGP-37157 enhances [Ca2+]m accumulation and restores mitochondrial NADH production in cells with elevated [Na+]i[9]. Mitochondria have several functions beyond ATP production, including acting as an intracellular Ca2+ buffering system, and we have shown that inhibition of [Ca2+]m efflux by CGP-37157 increases mitochondrial Ca2+ retention capacity and consequently decreases cytosolic Ca2+ ([Ca2+]c) cycling [8]: this effect might also attenuate glycoside-induced SR Ca2+ overload and arrhythmias.

In the present study, we investigated whether ouabain has adverse effects on mitochondrial energetics, redox status, and ROS balance, as well as whether CGP-37157 can prevent ouabain-induced mitochondrial dysfunction. Moreover, we also studied the role of mitochondrial dysfunction in ouabain toxicity by assessing the effects of CGP-37157 on ouabain-induced irregular [Ca2+]c cycling in isolated myocytes and on ouabain-induced arrhythmias in isolated perfused hearts. Finally, we show that CGP-37157 treatment has a beneficial effect to decrease in vivo arrhythmias in ouabain-treated guinea pigs.

Section snippets

Animal

Hartley guinea pigs (250–300 g; HillTop Lab Animals) were housed in an animal facility at the Johns Hopkins University. This study conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Johns Hopkins Animal Care and Use Committee.

Cell isolation

Guinea pig ventricular myocytes were isolated by enzymatic digestion as described previously [11]. Cells were suspended in Dulbecco's Modification

Effects of ouabain on [Na+]i

Application of ouabain to isolated myocytes at rest or with 1-Hz stimulation elevated [Na+]i monotonically in a dose-dependent way (Fig. 1). One-hertz stimulation increased the rate of [Na+]i accumulation slightly (Fig. 1). To optimize the conditions of our study so that [Na+]i could be elevated efficiently, but with acceptable toxicity during the protocol, 1 μM ouabain was used in the following isolated myocyte experiments.

Effects on [Ca2+]m and mitochondrial NADH during increased work

In the presence of 100 nM isoproterenol, 1-Hz stimulation induced [Ca2+]m

Discussion

This study is the first to show that ouabain has adverse effects on mitochondrial function and that the consequent mitochondrial dysfunction contributes to the toxicity of ouabain. As we hypothesized, ouabain treatment impairs NADH production in isolated myocytes as a consequence of cytosolic Na+ loading, and this effect was prevented by CGP-37157. In isolated myocytes, CGP-37157 was shown to (i) enhance mitochondrial Ca2+ uptake in the presence of ouabain, (ii) decrease cytosolic diastolic Ca2+

Acknowledgments

This work was supported by NIH Grants P01 HL081427 and R01 HL101235 (BO'R).

References (33)

  • S.A. Hunt et al.

    ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society

    Circulation

    (2005)
  • S.H. Rahimtoola

    Digitalis therapy for patients in clinical heart failure

    Circulation

    (2004)
  • M. Gheorghiade et al.

    Digoxin in the management of cardiovascular disorders

    Circulation

    (2004)
  • C. Alicandri et al.

    Captopril versus digoxin in mild-moderate chronic heart failure: a crossover study

    J. Cardiovasc. Pharmacol.

    (1987)
  • C. Maack et al.

    Elevated cytosolic Na+ decreases mitochondrial Ca2+ uptake during excitation–contraction coupling and impairs energetic adaptation in cardiac myocytes

    Circ. Res.

    (2006)
  • T. Liu et al.

    Enhancing mitochondrial Ca2+ uptake in myocytes from failing hearts restores energy supply and demand matching

    Circ. Res.

    (2008)
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