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European Journal of Heart Failure 2007 9(11):1086-1094; doi:10.1016/j.ejheart.2007.08.004
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© 2007 European Society of Cardiology

Mechanistic insight into the functional and toxic effects of Strophanthidin in the failing human myocardium

Dirk von Lewinskia,b, Egbert Bispinga,b, Andreas Elgnera, Jens Kockskämpera,b and Burkert Pieskeb,*

a Abteilung Kardiologie und Pneumologie, Georg-August-Universität Göttingen Robert-Koch-Str. 40, 37075 Göttingen, Germany
b Abteilung Kardiologie, Medizinische Universität Graz Auenbruggerplatz 15, 8036 Graz, Austria

* Corresponding author. Tel.: +43 385 2544; fax: +43 385 3733. E-mail address: burkert.pieske{at}meduni-graz.at


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Cardiac glycosides are characterized by a narrow therapeutic range with Ca2+-overload and arrhythmias occurring at higher concentrations. Data on cardiac glycosides in isolated failing human myocardium are scarce and the frequency-dependent actions and toxicity of Strophanthidin have not yet been characterized.

Aims: To determine inotropic responses and toxicity of Strophanthidin in failing human myocardium.

Methods and results: Experiments were performed in trabeculae from 64 end-stage failing hearts. Developed force, and intracellular [Ca2+]i and [Na+]i were recorded with Strophanthidin (0.01 to 1 µmol/L; 37°C, 1 Hz) and compared to interventions with distinct mechanisms of action (elevated [Ca2+]o, Isoproterenol, and EMD57033). The effects of Strophanthidin on force–frequency behaviour were also assessed.

Strophanthidin exerted concentration-dependent positive inotropic effects. These were paralleled by increases in intracellular [Na+] as well as increasing [Ca2+]i-transients and SR-Ca2+-load. At high concentrations (>0.5 µmol/L), Strophanthidin caused afterglimmers and aftercontractions, with declining developed force despite further increasing [Ca2+]i-transients. The force–frequency-relationship and diastolic function at higher pacing rates was worsened by Strophanthidin in a concentration-dependent manner.

Conclusions: Strophanthidin toxicity was dependent on concentration, calcium load, beating rate and β-adrenergic receptor activation. Our data support the view that low doses, heart rate control and additional β-adrenergic receptor blockade are essential in the use of cardiac glycosides in heart failure.

Key Words: Cardiac glycosides • Human myocardium • Calcium • Contractile function • Arrhythmias

Received June 13, 2007; Accepted August 22, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac glycosides are recommended in the guidelines for chronic heart failure [1] and for acute heart failure in patients with atrial tachyarrhythmias for heart rate control [2]. The inotropic effect of cardiac glycosides mainly results from Na+/K+-ATPase inhibition [3], a protein with reduced expression in human heart failure [4,5]. In rabbit myocardium, inhibition of Na+/K+-ATPase results in a consecutive increase in [Na+]i causing a net influx of Ca2+ via the Na+/Ca2+-exchanger, thus increasing the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum (SR) [6]. However, additional targets for cardiac glycosides to increase inotropy, such as ryanodine receptors or sodium channels, have been identified [7-9].

In clinical practice, cardiac glycosides are characterized by a narrow therapeutic range. In the large DIG-trial, cardiac glycoside therapy reduced the rate of hospitalisations but did not affect total mortality in patients with symptomatic heart failure and sinus rhythm [9]. This was confirmed in a recent post hoc-analysis [10]. However, in another post hoc analysis "high-normal" serum levels of digoxin (>1 ng/mL) were associated with increased mortality, possibly due to lethal ventricular arrhythmias [11]. While the mechanism of arrhythmogenesis was not elucidated, it may well have resulted from glycoside-induced Ca2+-overload of the cardiac myocytes.

Since cardiac glycosides are typically used in patients with heart failure, we directly tested the concentration-dependent effects of Strophanthidin on intracellular Na+- and Ca2+-handling, systolic and diastolic function, as well as arrhythmic extra contractions in isolated end-stage failing human myocardium.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Experiments were performed in muscle strips obtained from 64 end-stage failing hearts (30 with ischaemic and 34 with dilated aetiology; 49 male and 15 female) and one non-failing donor heart. The mean age of patients was 53.8±2.0 years; ejection fraction was 22.4±1.3% and cardiac index 2.2±0.1 L/min1/m2. Premedication consisted of ACE-inhibitors or AT1-blockers in 43 patients, beta-blockers in 15 patients, cardiac glycosides in 41 patients, and diuretics in 53 patients. The study protocol was approved by the local ethics committee and all patients gave informed consent.

2.1. Muscle strip preparation
Trabeculae were dissected from the left or right ventricle, connected to an isometric force transducer and superfused with Tyrode's solution of the following composition (in mmol/L): Na+ 152, K+ 3.6, Cl– 135, HCO3– 25, Mg2+ 0.6, H2PO4– 1.3, SO42– 0.6, Ca2+ 2.5, glucose 11.2 and 5 I.U. insulin/L equilibrated with carbogen to a pH of 7.4. Muscles were electrically stimulated (1 Hz, 37 °C), and gradually stretched to optimum preload, i.e., the muscle lengths at which maximum twitch force amplitude was obtained. At this time point (baseline value) diastolic tension was 3.2±0.5 mN/mm2. Alterations in diastolic tension were related to the baseline value.

2.2. Concentration response curves
We performed cumulative concentration-response curves for Strophanthidin (0.01-1 µmol/L), Isoproterenol (0.001-10 µmol/L) and [Ca2+]o (2.5-15 mmol/L). Due to the relatively long time taken to reach steady-state (~20 min), only 3 concentrations of Strophanthidin were tested in each muscle strip.

2.3. Aequorin measurements
The Ca2+-regulated bioluminescent photoprotein aequorin was macroinjected as described previously [12]. Aequorin light emission was detected using a photomultiplier, which was mounted just above the glass cuvette containing the muscle.

2.4. Rapid cooling contractures
Rapid cooling contractures (RCCs) were elicited by a rapid decrease in the temperature of the muscle chamber from 37 °C to 1 °C by switching from a warm to a cold solution as previously described [13]. The resulting cooling contracture is an index for SR Ca2+-content.

2.5. Sodium measurements
Intracellular sodium concentration was measured as described previously [14]. Muscles were incubated with the Na+ indicator SBFI (35 µmol/L) for 180 min. Isometric twitches were evoked (0.2 Hz) in Tyrode's solution at 30 °C. Photobleaching of the dye was minimized by attenuating the intensity of the excitation light by neutral density filter and by limiting fluorescence recording to intervals of ~20 s every 2 min.

2.6. Drugs
Strophanthidin (Sigma) was dissolved in ethanol to create a stock solution of 10 mmol/L, this was further diluted in Aqua dest. for the experiments. Isoproterenol was dissolved in 2 mL Tyrode's solution containing 4 µL 1N HCl and 0.1 mg ascorbic acid to prevent oxidation of the drug. EMD57033 (Merck, Darmstadt, Germany) was dissolved in DMSO to create a stock solution of 10 mmol/L and added to the bathing solution.

2.7. Statistical analysis
Data are expressed as mean±SEM. Differences were compared by paired Student's-t-test or two-way repeated measures ANOVA followed by Student-Newman-Keuls test when appropriate. Statistical significance was taken as p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effects of Strophanthidin on contractile function and [Na+]i
Elevation of [Na+]i caused by inhibition of Na+/K+-ATPase is thought to underlie the positive inotropic effect of cardiac glycosides, but this effect has never been directly assessed in the failing human myocardium. Fig. 1A shows an original recording of isometric force (top) and [Na+]i (bottom). After the addition of 0.5 µmol/L Strophanthidin, developed force increased from 3.7 mN/mm2 to a maximum of 7.2 mN/mm2 after 20 min. [Na+]i increased in parallel by 5.9 mmol/L after 10 min and 8.8 mmol/L at maximum developed force. Average results from a total of 5 muscle strips from 4 failing hearts are presented in Fig. 1B. There was a linear parallel increase in developed force and [Na+]i. Within 10 min, developed force increased to 131.1±6.3% of the pre-drug value and [Na+]i rose by 7.44±1.57 mmol/L (both p<0.01).


Figure 01
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  		Fig. 1 A: Original recording of the effect of 0.5 µmol/L Strophanthidin on isometric twitch force (top) and [Na+]i (bottom) in a ventricular muscle strip from an end-stage failing human heart. Addition of Strophanthidin (bar on top) to the bath solution caused a time-dependent increase in twitch force and [Na+]i. B: Effects of Strophanthidin (0.5 µmol/L) on developed force (black circles) and changes in [Na+]i (green squares). Average data obtained from 5 muscle strips isolated from 4 failing hearts. #,*=p<0.05 vs. baseline; ##,**=p<0.01 vs. baseline. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
3.2. Effects of Strophanthidin on [Ca2+]i handling
The elevation of [Na+]i was expected to result in increased [Ca2+]i-transients via modulation of NCX activity and an associated gain of SR Ca2+-load. To test this hypothesis [Ca2+]i-transients were recorded in aequorin loaded muscle strips. At 0.3 µmol/L Strophanthidin a positive inotropic effect with a parallel increase in aequorin light signal was recorded. Increasing Strophanthidin to 1 µmol/L increased diastolic tension as well as diastolic aequorin light signal, afterglimmers also occurred indicating diastolic dysfunction possibly related to diastolic [Ca2+]i-overload. Due to the higher diastolic tension, developed force was slightly decreased (Fig. 2A, right) despite a further increase in [Ca2+]i-transient amplitude. [Ca2+]i-dependence of Strophanthidin induced inotropy at submaximal concentrations was also demonstrated in averaged original tracings of 10 single twitches of a muscle strip at baseline and after administration of 0.5 µmol/L Strophanthidin. Although developed force and aequorin light signal markedly increased, the total duration of the twitch and Ca2+-signals were not altered.


Figure 02
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  		Fig. 2 Original recordings of the effects of 0.3 µmol/L and 1 µmol/L Strophanthidin on aequorin light signals and twitch tension. At stable basal conditions 0.3 µmol/L Strophanthidin was added to the bathing solution (bar on top). After mechanical stabilisation Strophanthidin was increased to 1 µmol/L. Mechanical stabilisation occurred within 20 min. Diastolic tension and relaxation kinetics were only affected at high (1 µmol/L) concentrations of Strophanthidin.

 
The positive inotropic effect of Strophanthidin was concentration dependent, reaching a maximum at 0.5 µmol/L (Fig. 3A and B). At this concentration, developed force increased to 206.3±28.3% of the baseline value in ischaemic cardiomyopathy (ICM) and to 190.4±19.1% of the baseline value in dilated cardiomyopathy (DCM). The corresponding increases in aequorin light signal were to 161.1±12.1% (ICM) and 182.7±18.4% (DCM) of baseline values, respectively (all p<0.05). Developed force after Strophanthidin in 3 muscle strips from a non-failing heart is shown for comparison in both graphs; maximum force was 204% of the baseline value at 0.5 µmol/L Strophanthidin. Pretreatment of patients with cardiac glycosides did not affect the positive inotropic response or diastolic function of the muscle strips. We also tested the effect of Strophanthidin on SR Ca2+-content in failing human cardiac muscle, by assessing developed twitch force and rapid cooling contractures. The results are shown in Fig. 3C. It can be seen that the concentration-dependent positive inotropic effect of Strophanthidin was paralleled by an increase in the amplitude of the rapid cooling contracture. These data suggest that increased SR Ca2+-load underlies increased intracellular Ca2+-transients (and possibly arrhythmias) after Na+-pump-inhibition.


Figure 03
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  		Fig. 3 Concentration-response curves for Strophanthidin (0.01-1 µmol/L). Effect on developed force (filled circles) and aequorin light signal (open circles) in muscle strips from (A) ischaemic cardiomyopathy (ICM) and (B) dilated cardiomyopathy (DCM). For comparison developed force of 3 muscle strips of a non-failing heart (grey squares). *=p<0.05 vs. baseline. (C) Concentration-response curves for Strophanthidin (0.01-1 µmol/L). Effect on developed force (filled circles) and rapid cooling contractures (RCC; open triangles) in 5 muscle strips from 4 failing human hearts. *=p<0.05 vs. baseline.

 
3.3. Effects of Strophanthidin on arrhythmic events
In the high concentration range (i.e., at 0.5 and 1 µmol/L) ~50% of muscle strips developed arrhythmias, which were preceded by afterglimmers and aftercontractions. Most muscle strips without arrhythmias showed a biphasic force response to Strophanthidin: twitch force amplitude increased up to a concentration of 0.5 µmol/L. At 1.0 µmol/L, however, developed force declined and diastolic tension increased, this was associated with further increases in Ca2+-transients (Fig. 3 and Table 1).


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  		Table 1 Influence of Isoproterenol and [Ca2+]o on twitch and light signal kinetics

 
Strophanthidin toxicity occurred at lower concentrations in the presence of β-adrenergic stimulation (Fig. 4). 0.3 µmol/L Strophanthidin exerted a typical positive inotropic effect (increase in twitch tension by ~50% within ~20 min) without an increase in diastolic tension or the occurrence of aftercontractions. After additional application of 0.1 µmol/L Isoproterenol, however, aftercontractions (AC) and afterglimmers (AG) occurred, which may indicate Ca2+-overload. Isoproterenol alone, despite a comparable increase in Ca2+-transients, did not induce ACs or AGs under our experimental conditions.


Figure 04
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  		Fig. 4 A: Original recording of the effect of 0.3 µmol/L Strophanthidin (red bar on top) followed by 0.1 µmol/L Isoproterenol (yellow bar on top), on aequorin light signal and isometric twitch tension. Strophanthidin and Isoproterenol increased twitch force without affecting diastolic function. Afterglimmers (AG) and aftercontractions (AC) occurred after Isoproterenol probably as a result of substantial Ca2+-overload. B: Original recording of 0.1 µmol/L Isoproterenol (yellow bar on top) on aequorin light signal and isometric twitch tension. Without exposure to Strophanthidin, no afterglimmers or aftercontractions occurred. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

 
3.4. Subcellular mechanisms of action of Strophanthidin
To characterize the mechanisms of action of Strophanthidin more precisely, we directly compared the relationship between the increase in twitch force and the amplitude of the aequorin light signal ({Delta}F/{Delta}L) at increasing concentrations of the Na+-pump inhibitor Strophanthidin, the β-adrenoceptor agonist Isoproterenol, the myofilament Ca2+-sensitizer EMD57033 and [Ca2+]o. The resulting {Delta}F/{Delta}L-curves are plotted in Fig. 5. The {Delta}F/{Delta}L-ratio for increasing concentrations of Strophanthidin and [Ca2+]o run in parallel up to 0.5 µmol/L Strophanthidin where comparable increases in F and L are induced with [Ca2+]o 5.6 mmol/L. At this point the {Delta}F/{Delta}L-ratio is 1.3±0.3 for Strophanthidin and 1.3±0.2 for [Ca2+]o. At the highest concentration of Strophanthidin (1 µmol/L), the {Delta}F/{Delta}L-ratio-curve decreases due to declining force and further increasing Ca2+-transients ({Delta}F/{Delta}L-ratio: 0.8±0.2). This indicates impaired Ca2+-utilisation at high concentrations of Strophanthidin in the failing human myocardium.


Figure 05
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  		Fig. 5 Concentration-response curves for Isoproterenol (n=14; yellow circles), Strophanthidin (n=17; red circles), [Ca2+]o (n=16; blue circles), and a single concentration (n=7; 10 µmol/L) of EMD 57033 (black circles). Increases in developed force ({Delta}F) and aequorin light signal ({Delta}L) are plotted. The steepness of the curves (in comparison with the [Ca2+]o curve as reference) reflects Ca2+-sensitizing or -desensitizing effects. The curves for Strophanthidin and [Ca2+]o run in parallel. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

 
In comparison, Isoproterenol significantly reduced the {Delta}F/{Delta}L-ratio (0.3±0.05 at 10 µmol/L; p<0.05 vs. baseline value) and resulted in enhanced relaxation. This is the characteristic pattern of cAMP-dependent interventions with overproportional increases in [Ca2+]i [15]. In contrast, EMD57033 (10 µmol/L) increased the {Delta}F/{Delta}L-ratio due to a significant increase in twitch force (331.6±66%; p<0.05) with no increase in aequorin light signals, time relaxation kinetics became slower which is typical for a Ca2+-sensitizing agent. In contrast, twitch and aequorin light signal kinetics were not altered with Strophanthidin or increases in [Ca2+]o (Table 2).


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  		Table 2 Influence of Strophanthidin on kinetics of twitch (RT) and aequorin light (RL) signals and diastolic tension

 
These experiments support the notion that Strophanthidin, in contrast to Isoproterenol, exerts its positive inotropic effects at non-toxic concentrations by a cAMP-independent increase in intracellular Ca2+-transients and these effects are purely Ca2+-dependent without changes in myofilament Ca2+-sensitivity.

3.5. Frequency-dependent adverse effects of Strophanthidin on contractile function
To test the frequency-dependence of Strophanthidin toxicity we compared force-frequency relations (FFR) before and after incubation with low (0.03 µmol/L; n=4) or submaximal (0.3 µmol/L; n=7) concentrations of Strophanthidin. Muscle strips from failing hearts were characterized by a typical negative FFR (Fig. 6 left) with decreases in developed force at 3 Hz to 49±5% of baseline value before 0.03 µmol/L Strophanthidin and 49±12% before 0.3 µmol/L Strophanthidin (Fig. 6) respectively (both p<0.05). At 0.5 Hz, Strophanthidin increased developed force by 15±4% (0.03 µmol/L) and 51±12% (0.3 µmol/L), respectively (both p<0.05). After complete mechanical stabilisation of the muscle strips, a second FFR was tested. At 0.03 µmol/L Strophanthidin, the FFR was only slightly shifted towards a steeper decline (at 3 Hz 42±7% of baseline; p<0.05 vs. baseline) and none of the muscle strips developed arrhythmias. However, 0.3 µmol/L Strophanthidin significantly worsened the FFR (at 3 Hz developed force was 15±4% of baseline; p<0.05 vs. baseline and p<0.05 vs. 3 Hz without Strophanthidin) and the muscle strips were characterized by increasing diastolic tension and frequent arrhythmias.


Figure 06
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  		Fig. 6 Left: Baseline FFR. Right: FFR in the presence of a high concentration of Strophanthidin (0.3 µmol/L). Developed systolic force: black triangles; diastolic tension: grey triangles. *=p<0.05 vs. baseline.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This is the first report on rate dependent cardiac glycoside toxicity in isolated human myocardium from end-stage failing hearts. The results show that 1) Strophanthidin exerts concentration-dependent positive inotropic, but also toxic effects (arrhythmias, diastolic dysfunction) at concentrations >0.5 µmol/L; 2) The functional effects are Na+- and Ca2+-dependent; 3) Strophanthidin facilitates Ca2+-overload compared to β-adrenergic stimulation; 4) Increasing stimulation rates shift Strophanthidin toxicity towards lower concentrations.

4.1. Mechanism of action
Although cardiac glycosides have been used in clinical practice for more than 200 years there is still an ongoing debate about their mode of action. The main target of cardiac glycosides is Na+/K+-ATPase. Inhibition of this ion transporter results in an accumulation of [Na+]i, which shifts the activity of Na+/Ca2+-exchanger towards less forward and more reverse mode and thus increases [Ca2+]i [16]. Recent data demonstrate that phospholemman regulates Na+/K+-ATPase-activity [17]. Unphosphorylated phospholemman inhibits Na+/K+-ATPase activity, whereas phosphorylation of phospholemman results in reduced inhibition of the Na+-pump and therefore increased activity of Na+/K+-ATPase analogous to the regulation of SERCA by phospholamban.

Additional mechanisms of action of cardiac glycosides have been described. Isenberg [7] reported positive inotropic effects of cardiac glycosides even in the absence of sodium or in the presence of digoxin-specific antibodies. Targets that may contribute to these functional effects are the ryanodine receptor [9] and the sarcolemmal tetrodotoxin-sensitive Na+-channels [18] which may conduct Ca2+-ions in the presence of cardiac glycosides. Nevertheless, "slip mode"-conductance of Ca2+-ions via Na+-channels has recently been challenged [19] and may not be of major importance for the inotropic effects of glycosides in the failing human myocardium. In human atrial myocardium, glycosides may also increase myofilament Ca2+-sensitivity, but the mechanism remains speculative [20]. Therefore, the "classic" pathway via Na+/K+-ATPase-inhibition and Ca2+-influx via the Na+/Ca2+-exchanger is still the most important mechanism for both inotropic and toxic effects of cardiac glycosides. In fact, we identified an increase in [Na+]i as a major mechanism of action of Strophanthidin in the failing human myocardium.

4.2. Intracellular Na+- and Ca2+-handling
In failing human cardiac muscle, Strophanthidin had a narrow concentration range for non-toxic positive inotropic effects. There were no significant inotropic effects at concentrations <0.1 µmol/L, whereas toxic effects with substantial diastolic dysfunction, reduced twitch force and arrhythmic extra contractions were observed at concentrations >0.5 µmol/L. Concentration-dependent positive inotropic effects of Strophanthidin without diastolic dysfunction were observed at concentrations between 0.1 and 0.5 µmol/L. At 0.5 µmol/L (and a relatively low stimulation rate of 1 Hz), developed force was increased by ~100% of the basal value without delayed relaxation time or aftercontractions. These effects as well as the toxic effects at higher concentrations were independent of cardiac glycoside premedication of patients prior to transplantation (data not shown).

An increase in [Na+]i and [Ca2+]i as the underlying mechanism for the inotropic effect of Strophanthidin has been demonstrated in a number of mammalian species. Since basal [Na+]i is elevated [14] and the Na+/Ca2+-exchanger is upregulated in failing human hearts we assessed the effects of Strophanthidin on intracellular Na+- and Ca2+-handling in failing human cardiac muscle. We found that intracellular Na+ increased in the presence of Strophanthidin. This increase in [Na+]i was associated with an increase in systolic intracellular Ca2+-transients and SR Ca2+-load, most probably as a result of reverse-mode NCX. We did not compare failing to non-failing myocardium in this study and the data in non-failing myocardium should be interpreted with caution since only one control heart was available, but elevated basal [Na+]i and upregulated NCX may both contribute to a higher functional responsiveness of failing human heart to cardiac glycosides.

Inotropic interventions may be associated with increased, unchanged, or decreased myofilament Ca2+-sensitivity [21]. Typically, an intervention that results in increased myofilament Ca2+-responsiveness prolongs twitch kinetics, while an intervention that reduces Ca2+myofilament sensitivity has the opposite effect. In the present study, the inotropic effects of Strophanthidin were not associated with altered twitch kinetics even at high concentrations. Furthermore, Ca2+-sensitizing inotropic interventions are characterized by underproportional increases in intracellular Ca2+-transients, while Ca2+-desensitizing interventions show overproportional increases [21]. Strophanthidin, as compared to an increase in the extracellular Ca2+-concentration as a reference intervention with no effects on myofilament sensitivity, showed comparable increases in Ca2+-transients relative to force increase. In contrast, Isoproterenol exerted overproportional increases in Ca2+-transients whereas EMD57033 did not result in any Ca2+-increase. These comparative data suggest that submaximal concentrations of Strophanthidin exert functional effects solely by elevating [Ca2+]i without affecting the Ca2+-sensitivity of the myofilaments.

This finding is in contrast to a recent paper by Hambachian and coworkers, who observed increased myofilament Ca2+-sensitivity in the presence of ouabain in skinned human atrial muscle [20]. Besides differences in atrial vs. ventricular cardiac muscle, a putative Ca2+-sensitizing effect may be of minor relevance even in their model under more physiological conditions, because neither twitch nor Ca2+-transient decay times were altered with ouabain [20].

4.3. Differences in glycoside effects in non-failing vs. failing heart
In human heart failure, Na+/K+-ATPase is downregulated by ~40% [4,5] and this might enhance Na+/K+-ATPase-inhibitor toxicity in the failing myocardium. This would support the theory that this downregulation correlates with ejection fraction [4]. However, expression of the regulating protein phospholemman is also reduced, whereas intrinsic phospholemman activity is increased [22] in human heart failure. Therefore, functional effects are difficult to predict. Besides Na+/K+-ATPase, recent findings demonstrate increased intracellular Na+-concentrations [14], an enhanced sensitivity towards Na+-channel activation [23], "leaky" SR Ca2+-release channels [24] and a reduced SERCA2a activity [25]. Due to the reduced driving force of the Na+-gradient [14] and blunted SR function, cytosolic Ca2+-elimination is impaired in the failing human myocardium. Although this may also render the failing human heart particularly sensitive to the toxic effects of cardiac glycosides, functional effects of cardiac glycosides have been shown to be as effective in isolated failing and non-failing human myocardium [23,26] and this in line with the data on non-failing myocardium shown in Fig. 3. Therefore, potential toxicity-increasing mechanisms such as increased [Na+]i, downregulated Na+/K+-ATPase and altered Ca2+-handling seem to be compensated by other mechanisms, probably including phospholemman activity.

4.4. Strophanthidin toxicity
Heart failure is associated with increased sympathetic tone, high heart rates, and eventually atrial fibrillation. In these situations, patients are often treated with cardiac glycosides. However, both sympathetic tone as well as high heart rates may shift the functional effects of cardiac glycosides towards toxicity. Indeed, in the present study, increasing Strophanthidin concentrations above a narrow inotropic range resulted in frequent arrhythmias and elevated diastolic tension.

High catecholamine plasma-levels may further potentiate glycoside dependent arrhythmogenesis due to an increase in intracellular Ca2+-load and enhanced SERCA-activity. In the present study, Isoproterenol induced afterglimmers and aftercontractions in aequorin loaded trabeculae only after preincubation with per se non-toxic concentrations of Strophanthidin. β-adrenoceptor-stimulation increases SR-Ca2+-load and RyR2 Ca2+-release via PKA-dependent phosphorylation processes [27]. Spontaneous Ca2+-release from the SR as a probable mechanism for afterglimmers and aftercontractions is dependent on SR Ca2+-load and RyR2 release properties. Therefore, β-adrenergic stimulation amplifies the proarrhythmogenic potential of Strophanthidin and shifts the threshold for arrhythmias towards lower Strophanthidin concentrations. This effect can be observed although diastolic relaxation is improved with isoproterenol (Table 1) and despite the fact that isoproterenol increases Na+/K+-ATPase activity via PKA-mediated phosphlemman-phosphorylation [17].

Failing human myocardium is characterized by an inverse force-frequency relation due to dysfunctional intracellular Ca2+-handling. At higher beating rates, diastole markedly shortens and consequently time to eliminate Ca2+ from the cytosol decreases. Therefore, vulnerability for cytosolic Ca2+-overload and diastolic dysfunction is augmented at higher pacing rates. Cardiac glycosides increase intracellular Ca2+-turnover and SR Ca2+-load without direct effects on cytosolic Ca2+-elimination processes [28]. Consistent with this notion, we observed progressive diastolic dysfunction at increasing pacing rates in the presence of "non-toxic" concentrations of Strophanthidin.

Recent post-hoc analysis of the DIG trial [10,11] has demonstrated beneficial effects of low doses of digoxin whereas higher doses were without beneficial effects or even associated with higher mortality. There is no subgroup analysis on the effect of cardiac glycosides in patients co-treated with β-blockers. However, the data from our study indicate that β-blockers may prevent triggered arrhythmias in patients on cardiac glycosides because they reduce SR Ca2+-overload and PKA-dependent RyR2-hyperphosphorylation [29]. In addition, heart rate control with longer duration of diastole facilitates diastolic Ca2+-elimination from the cytosol and therefore prevents glycoside-associated diastolic dysfunction. The latter may be of specific relevance in heart failure patients treated for atrial tachyarrhythmias.

In some muscle strips, arrhythmias occurred before diastolic tension increased, whereas others developed contracture without the occurrence of arrhythmias. These differences might be based on different levels of Na+/Ca2+-exchanger and SERCA2a expression which vary to a great extent in failing human myocardium [30] as well as on the "leakiness" of the ryanodine receptor. It can be speculated that muscle strips with high Na+/Ca2+-exchange and low SERCA2a expression tend to accumulate Ca2+ within the cytosol and develop contracture, whereas muscle strips with higher SERCA2a-levels but leaky ryanodine receptors may be more susceptible for triggered arrhythmias. Therefore, the prevailing toxic effect of cardiac glycosides (diastolic dysfunction vs. triggered arrhythmias) may depend on the clinical situation (drug dose, heart rate), but also on the individual phenotype.

In conclusion, we have demonstrated, for the first time in failing human myocardium, a concentration and rate dependent increase in cardiac glycoside toxicity. This is of clinical relevance and emphasizes the importance of low plasma concentrations and the additional relevance of heart rate control in heart failure patients treated with cardiac glycosides.


   Acknowledgement
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Pi-414/1-3) to Burkert Pieske.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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