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Cardiovascular Research 2000 45(4):913-924; doi:10.1016/S0008-6363(99)00387-9
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Copyright © 2000, European Society of Cardiology

Gingerol, isoproterenol and ouabain normalize impaired post-rest behavior but not force–frequency relation in failing human myocardium

Lars S Maiera, Christiane Schwana, Wolfgang Schillingera, Kazutomo Minamib, Ulrich Schüttb and Burkert Pieskea,*

aGeorg-August-Universität Göttingen, Abteilung Kardiologie und Pneumologie, Zentrum Innere Medizin, Robert-Koch-Str. 40, 37075 Göttingen, Germany
bHerzzentrum Nordrhein-Westfalen, Bad Oeynhausen, Germany

* Corresponding author. Tel.: +49-551-39-8925; fax: +49-551-39-8925 pieske{at}med.uni-goettingen.de

Received 30 July 1999; accepted 20 October 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Rest- and stimulation frequency-dependent potentiation of contractile force is blunted in failing human myocardium. These alterations have been related to reduced sarcoplasmic reticulum (SR) Ca2+-reuptake and enhanced transsarcolemmal Ca2+-elimination by Na+/Ca2+-exchange. We investigated whether inotropic interventions that enhance SR Ca2+-uptake, or reduce Ca2+-elimination by Na+/Ca2+-exchange, normalize impaired post-rest and force–frequency behavior in left ventricular muscle strips from failing human hearts. Methods: We tested the influence of [10]-gingerol which activates SR Ca2+-ATPase (10 µmol/l; n=13), and isoproterenol which activates cAMP-dependent pathways (0.01, 0.1, 1 µmol/l; n=40) on post-rest and force–frequency behavior. Ouabain which blocks Na+/K+-ATPase (0.03 µmol/l; n=16) was used to test the effects of inhibiting Ca2+-elimination by Na+/Ca2+-exchange. For comparison, the effects of blocking SR Ca2+-uptake by thapsigargin (10 µmol/l; n=14) were tested. In addition, Ca2+-uptake in myocardial homogenates was measured for gingerol (10 µmol/l; n=6). Results: Gingerol, isoproterenol (0.1, 1 µmol/l) and ouabain exerted significant positive inotropic effects under basal experimental conditions and normalized post-rest behavior. In contrast, force–frequency relation was only slightly improved by gingerol and isoproterenol (0.01 µmol/l). Ouabain and isoproterenol (1 µmol/l) further deteriorated force–frequency relation due to frequency-dependent significant increases in diastolic tension. Thapsigargin exerted negative inotropic effects and significantly deteriorated post-rest and force–frequency behavior. In addition, gingerol increased SR Ca2+-uptake significantly in myocardial homogenates. Conclusions: Inotropic interventions that stimulate SR Ca2+-ATPase or inhibit Na+/Ca2+-exchange normalize impaired post-rest behavior. Force–frequency behavior is only slightly improved by stimulation of SR Ca2+-ATPase but not by inhibition of Na+/Ca2+-exchange. This dissociation between post-rest and force–frequency behavior results from diastolic dysfunction at high stimulation rates.

KEYWORDS Contractile function; E–C coupling; Heart failure; Inotropic agents


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Potentiation of isometric twitch force after a rest interval (post-rest potentiation) is an index for sarcoplasmic reticulum (SR) Ca2+-uptake capacity [1]. In nonfailing human myocardium, post-rest potentiation of force can be seen up to rest intervals of several minutes with parallel increases in intracellular Ca2+-transients [2]. In contrast, in end-stage failing human myocardium post-rest potentiation of force is blunted at long rest intervals (post-rest decay) due to decreased intracellular Ca2+-transients [2]. In addition, a frequency-dependent increase in force in nonfailing human myocardium (positive force–frequency relation) is blunted in failing myocardium (negative force–frequency relation) [3,4], associated with a decline in intracellular Ca2+-transients [5]. Therefore, contractile dysfunction of failing human myocardium may be due to changes in intracellular Ca2+-handling possibly as a result of both decreased expression and function of SR Ca2+-ATPase [5–8], and increased expression and function of sarcolemmal Na+/Ca2+-exchanger [9–12]. This is further supported by the direct correlation between impaired force–frequency relation and the degree of reduced expression of SR Ca2+-ATPase [6]. In addition, it was observed that the ratio of Na+/Ca2+-exchanger to SR Ca2+-ATPase is considerably increased in failing myocardium [9]. Thus, the upregulated Na+/Ca2+-exchanger seems to eliminate more Ca2+ from the cytosol leading to a shift from intracellular to transsarcolemmal Ca2+-cycling [13].

Consequently, it was shown recently that reduced SR Ca2+-reuptake and increased Na+/Ca2+-exchange limits SR Ca2+-accumulation [14], leading to the observed negative force–frequency relation and post-rest decay in failing human myocardium [2,5].

We investigated whether pharmacological interventions which directly (gingerol) or indirectly (isoproterenol) stimulate SR Ca2+-uptake might normalize impaired post-rest and force–frequency behavior in failing human myocardium. Furthermore, the effects of ouabain which blocks Na+/K+-ATPase thereby preventing Ca2+-elimination by Na+/Ca2+-exchange, and the SR Ca2+-pump inhibitor thapsigargin were analyzed.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Myocardial tissue
Experiments were performed in left ventricular muscle strips obtained from 12 nonfailing donor hearts that could not be transplanted for technical reasons (mean age 47±5 years), and in 43 explanted end-stage failing hearts with ischemic (n=14) or dilated (n=29) cardiomyopathy (mean age 52±2 years) and a mean ejection fraction of 26±2%. Premedication of the patients usually consisted of digitalis glycosides (n=30), diuretics (n=27), ACE-inhibitors (n=27) and β-blockers (n=4). Individual premedication did not affect the results of this study. The study was approved by the ethical committee of the University of Freiburg and conforms with the principles outlined in the Declaration of Helsinki.

2.2 Muscle strip preparation
Isolation of muscle strips was carried out as described previously [5]. Briefly, after explantation, the hearts were washed in Krebs-Henseleit-buffer (KHB) containing (mmol/l): Na+ 152, K+ 3.6, Cl– 135, HCO3– 25, H2PO4– 1.3, Mg2+ 0.6, SO42– 0.6, Ca2+ 2.5, glucose 11.2 and insulin 10 IU/l. The KHB was continuously bubbled with carbogen (pH 7.4). During transportation and preparation, the hearts were stored in a cardioplegic KHB containing 30 mmol/l 2,3-butanedione monoxime (BDM). Thin ventricular muscle strips were excised for functional measurements and tied with loops of fine silk suture at both ends. Muscles were transferred to an organ chamber and attached between a force transducer and a fixed pin. After washout of the cardioplegic solution with standard KHB (without BDM) at 37°C, muscles were electrically stimulated at 1 Hz (voltage 20% above threshold, 5 ms duration) and stretched gradually to the length at which maximum twitch force was reached. Force was amplified and recorded on a strip-chart recorder. At the end of the experiment, muscle length and wet weight were measured. Cross-sectional area was determined by dividing wet weight by muscle length. Average muscle length was 4.9±0.6 mm and cross-sectional area was 0.40±0.06 mm2, without significant differences between subgroups.

2.3 Pharmacological interventions
Isoproterenol (0.01, 0.1, 1 µmol/l) and ouabain (0.03 µmol/l) were added to the KHB. To test for the effects of gingerol, one muscle strip (>10 mm length) was divided into two preparations of similar length. One preparation was incubated for 6 h with [10]-gingerol (10 µmol/l) in a modified KHB which additionally contained 0.5 vol% Cremophor to facilitate penetration of the compound. The other preparation was incubated for 6 h in the same KHB without gingerol (control). Similarly, one preparation was incubated in the KHB containing thapsigargin (10 µmol/l) and the other preparation without the compound. All compounds were purchased from Sigma Chemicals except for gingerol (Glaxo, France).

2.4 Experimental protocol for functional measurements
In order to investigate the influence of rest, rest intervals (1–240 s) were instituted from a basal stimulation rate of 1 Hz without and in the presence of one compound. Post-rest force is defined as the amplitude of the first twitch upon restimulation. It is compared to the amplitude of the steady-state force before rest. Force–frequency relations were tested by stepwise increasing stimulation rate (0.5–3 Hz) without and in the presence of one compound. At each stimulation frequency, steady-state contractile parameters of the isometric twitches were assessed and compared to the value at 0.5 Hz.

2.5 Calcium-uptake measurements
ATP-dependent, oxalate-facilitated Ca2+-uptake was measured as previously published [5]. Briefly, 60–80 mg of myocardium, which was frozen in liquid nitrogen at the time of explantation of the hearts, were thawed in 10 ml ice-cold solution containing 25 mmol/l imidazole (pH 7.0), minced, and homogenized. An aliquot of each sample was transferred into the uptake medium containing (mmol/l): imidazole 40, KCl 100, potassium oxalate 5, MgCl2 4.5, NaN3 10, Na2ATP 2.5, creatine phosphate 3, and creatine phosphokinase 2 IU/ml. For measuring the effect of gingerol on SR Ca2+-uptake, 10 µmol/l gingerol was present in the reaction medium and the mixture was preincubated for 15 min (37°C). The influence of cyclopiazonic acid (CPA) was determined for comparison by preincubating the assay-mixture for 3 min (37°C) in the presence of 10 µmol/l CPA. Specimens from the same hearts served as a control and underwent the procedure in parallel, without gingerol or CPA. After the preincubation period, the reaction was started by adding (µmol/l): CaCl2 25 (0.185 µCi 45Ca2+/ml) and EGTA 15.5, yielding a free Ca2+-concentration of 7 µM. After intervals of 0.5, 1, 1.5 and 2 min, 100 µl aliquots were filtered, washed with ice-cold solution containing (mmol/l): KCl 0.6, NaN3 5, and imidazole 20. Filters were analyzed in a liquid scintillation counter and Ca2+-uptake was calculated from the radioactivity as nmol 45Ca2+/g myocardium for the different time intervals. Ca2+-uptake rate was calculated from the slope of the linear regression analysis as nmol 45Ca2+/g myocardium/min. Each individual value represents the mean of three independent determinations.

2.6 Statistics
All data are expressed as mean±S.E.M. For functional measurements, statistical analysis was performed with two-way repeated measurements ANOVA followed by Student-Newman-Keuls test or Student's t-test where appropriate. Linearity of the Ca2+-uptake assay was checked by linear regression analysis. Comparisons of the influence of pharmacological interventions on Ca2+-uptake rate vs. control was checked using Student's t-test. Statistical significance was taken as P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Alterations in contractile behavior in heart failure
Fig. 1 summarizes post-rest (left) and force–frequency (right) contractile behavior in human nonfailing and failing myocardium. Nonfailing myocardium (n=12) showed a progressive potentiation of force after increasing rest intervals up to 240 s (by 123±24% vs. force at 1 Hz; P<0.05). In failing human myocardium (n=38), rest potentiation of force increased only at short rest intervals and then declined continuously with longer rest intervals up to 240 s (by 20±9%; P<0.05). Moreover, nonfailing myocardium (n=12) showed a frequency-dependent increase in force after increasing stimulation frequency to 3 Hz (by 51±19% vs. force at 0.5 Hz; P<0.05). In failing human myocardium (n=43), force declined continuously with higher stimulation frequencies to 3 Hz (by 31±6%; P<0.05).


Figure 1
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  		Fig. 1 Post-rest (left) and force–frequency (right) contractile behavior in human nonfailing ({circ},{square}: n=12) and end-stage failing (bullet: n=38, {blacksquare}: n=43) myocardium. *significantly different (P<0.05) to steady-state force, #significantly different (P<0.05) to nonfailing myocardium.

 
3.2 Effects of inotropic interventions on twitch force
Table 1 summarizes mean data of twitch force and time parameters for the different compounds at 1 Hz in failing human myocardium. Isoproterenol concentration-dependently increased force by 0.5±0.2 mN/mm2 (0.01 µmol/l; n=11; n.s.), 4.6±0.8 mN/mm2 (0.1 µmol/l; n=13; P<0.05) and by 9.2±1.5 mN/mm2 (1 µmol/l; n=16; P<0.05). Gingerol (10 µmol/l; n=13) increased force by 1.2±0.4 mN/mm2 (P<0.05), and ouabain (0.03 µmol/l; n=16) by 2.6±0.7 mN/mm2 (P<0.05). In contrast, thapsigargin (10 µmol/l; n=14) decreased force by 3.9±1.1 mN/mm2 (P<0.05). In addition, isoproterenol and gingerol significantly enhanced relaxation parameters of twitches. In contrast, ouabain did not change time parameters and thapsigargin significantly prolonged time parameters.


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  		Table 1 Influence of isoproterenol, gingerol, ouabain and thapsigargin on twitch force and time parametersa

 
3.3 Effects of inotropic interventions on post-rest behavior
Fig. 2 shows original recordings of the effects of gingerol, isoproterenol (1 µmol/l), ouabain and thapsigargin in failing human myocardium before and after 10 and 120 s rest. In Fig. 2A, a small post-rest potentiation of force can be seen after 10 s rest which is enhanced in the presence of gingerol. In contrast, at 120 s there is no post-rest potentiation for control, but a strong rest potentiation of force with gingerol. Similarly, isoproterenol (Fig. 2B) and ouabain (Fig. 2C) increased post-rest potentiation of force at 10 s and induced rest-potentiation of force at 120 s similar to nonfailing human myocardium [2,14]. In contrast, thapsigargin (Fig. 2D) induced rest decay of force already after 10 s, and severely suppressed force after 120 s rest.


Figure 2
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  		Fig. 2 Post-rest twitches in isolated muscles from failing human hearts (basal stimulation frequency 1 Hz). A): Without (control) and with gingerol. After 10 s rest (above) there is post-rest potentiation of force which is even pronounced at 120 s rest (below) with gingerol, whereas under control conditions there is post-rest decay of force. B): Without (control) and with isoproterenol (1 µmol/l). C): Without (control) and with ouabain. With isoproterenol and ouabain, post-rest potentiation further increases after 120 s in contrast to rest-decay under control conditions. D): Without (control) and with thapsigargin. After 10 s rest there is already post-rest decay of force with thapsigargin which is even pronounced at 120 s rest vs. control.

 
As can be seen from Fig. 3, average post-rest force without pharmacological interventions increased after short rest intervals but declined with longer rest intervals. However, all interventions induced a pronounced change in post-rest contractile behavior: gingerol (n=8) converted rest-decay into rest potentiation at all rest intervals. After 240 s rest, post-rest twitch force was 185±44% of the steady-state force at 1 Hz vs. 68±24% without gingerol (P<0.05). Similarly, isoproterenol (1 µmol/l; n=9) and ouabain (n=6) converted rest decay to rest potentiation of force. After 240 s rest, force was 175±13% in the presence of isoproterenol vs. 68±8% without isoproterenol (P<0.05). Additionally, after 480 s post-rest force in six muscles was tested and was still increased to 138±5% (P<0.05; not shown). With a lower concentration of isoproterenol (0.1 µmol/l; n=6; not shown), post-rest force increased to 159±19% vs. 65±13% without isoproterenol after 240 s rest (P<0.05). Similarly, ouabain significantly improved post-rest behavior: Before ouabain, twitch force at 240 s rest was 108±24%. However, in the presence of ouabain, post-rest contractile force was significantly increased after short rest intervals and remained stable even after 240 s rest (233±41%; P<0.05). In contrast, with thapsigargin (n=9) post-rest potentiation was completely abolished at short rest intervals and was 43±15% after 240 s rest (vs. 95±24% without thapsigargin; P<0.05). There were no significant differences in contractile behavior between non-ischemic and ischemic cardiomyopathies.


Figure 3
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  		Fig. 3 Mean rest-dependent changes in post-rest force in failing human myocardium for muscles with gingerol (n=8), isoproterenol (1 µmol/l; n=9), ouabain (n=6), and thapsigargin (n=9) ({circ}) vs. control without interventions (bullet). Values are normalized to steady-state force (1 Hz), *significantly different (P<0.05) to steady-state force, #significantly different (P<0.05) to control.

 
3.4 Effects of inotropic interventions on force–frequency relation
Mean values for the force–frequency relations without and in the presence of the inotropic interventions are presented in Fig. 4. Gingerol (n=5) slightly improved force–frequency behavior. Twitch force increased to 129±22% at 2 Hz and to 107±16% at 3 Hz (n.s) compared to the steady-state force at 0.5 Hz (without gingerol force decreased to 85±11% at 3 Hz). At a low concentration, isoproterenol (0.01 µmol/l; n=11) slightly improved force–frequency relation (the decline in force was prevented up to 2 Hz stimulation frequency). However, an intermediate concentration of isoproterenol (0.1 µmol/l; n=7; not shown) had no effects on force–frequency relation (force at 3 Hz was 39±6% with, vs. 49±10% without isoproterenol; n.s.), and a high concentration of isoproterenol (1 µmol/l; n=7; not shown) further impaired force–frequency behavior (force at 3 Hz was 39±7% vs. 55±12% without isoproterenol; P<0.05). Ouabain (n=10) significantly deteriorated force–frequency behavior. Twitch force continuously decreased and was at 3 Hz 44±6% of the basal force (vs. 89±16% without ouabain; P<0.05). Differences between ouabain and control values were significant at stimulation rates above 1 Hz. In addition, thapsigargin (n=5) further deteriorated force–frequency relation. Force decreased to maximally 39±9% at 3 Hz (vs. 66±10% without thapsigargin; P<0.05). At frequencies above 2 Hz there was a significant difference between thapsigargin and control. It should be noted that the variations in force–frequency behavior between the control groups were not statistically significant and there were no significant differences in contractile behavior between non-ischemic and ischemic cardiomyopathies.


Figure 4
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  		Fig. 4 Frequency-dependent changes in force in failing human myocardium. All values are normalized to steady-state force (0.5 Hz). Mean values for muscles with gingerol (n=5), isoproterenol (0.01 µmol/l; n=11), ouabain (n=10), and thapsigargin (n=5, all {circ}) vs. control without interventions (bullet), *significantly different (P<0.05) to steady-state force, #significant difference (P<0.05) between intervention and control.

 
Fig. 5 shows the effects of stimulation rate on diastolic tension (as change in mN/mm2 from baseline conditions at 3 Hz vs. 0.5 Hz). In the presence of gingerol, diastolic tension did not differ from control without the compound (+0.7±0.3 mN/mm2 with vs. +0.3±0.1 mN/mm2 without gingerol). However, isoproterenol at a low concentration (0.01 µmol/l) improved diastolic function at higher stimulation frequencies vs. control (+1.1±0.4 mN/mm2 with vs. +3.3±1.3 mN/mm2 without isoproterenol; P<0.05). Ouabain impaired diastolic function at high stimulation rates (+3.1±1.0 mN/mm2 with, vs. 0.7±0.3 mN/mm2 without ouabain; P<0.05). In addition, thapsigargin increased diastolic tension significantly at 3 Hz compared to 0.5 Hz, whereas under control conditions diastolic tension did not increase significantly (+3.4±0.5 mN/mm2 with vs. +2.7±1.0 mN/mm2 without thapsigargin). It should be noted that as with active twitch tension, there was some variation in diastolic force–frequency behavior between control groups. This variability has been previously observed and may partly be related to differences in Na+/Ca2+-exchanger protein expression [9].


Figure 5
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  		Fig. 5 Frequency-dependent changes in diastolic force in failing human myocardium from the data from Fig. 4. Gingerol, isoproterenol, ouabain, and thapsigargin ({circ}) vs. control without interventions (bullet), *significantly different (P<0.05) to steady-state force, #significantly different (P<0.05) to control.

 
3.5 Effects of gingerol on SR calcium-uptake
Fig. 6 shows SR Ca2+-uptake data for gingerol in left ventricular homogenates (n=6). On the left, the linearity of Ca2+-uptake over a period of 2 min in the control group and in myocardial homogenates preincubated with 10 µmol/l [10]-gingerol is demonstrated. On the right, Ca2+-uptake rate per min is calculated from the slope of the linear regression analysis. Preincubation with gingerol resulted in a significant increase in Ca2+-uptake rate by 97% vs. control (704±136 vs. 357±115 nmol Ca2+/g myocardium/min). In contrast, preincubation with 10 µmol/l CPA (n=3) resulted in a significant decrease in Ca2+-uptake rate by 90% (not shown).


Figure 6
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  		Fig. 6 Ca2+-uptake in homogenates from failing human myocardium (n=6). Left: Linearity of Ca2+-uptake for control (r=0.99, P=0.007) and in the presence of 10 µmol/l gingerol (r=0.98, P=0.02). Right: Ca2+-uptake rate per min calculated from the slope of the linear regression analysis. Preincubation in the presence of 10 µmol/l gingerol resulted in an increase of Ca2+-uptake rate by 97% (P=0.0003) vs. control.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study shows that (1) altered post-rest behavior of twitch force in end-stage failing myocardium can be normalized with direct (gingerol) and indirect (isoproterenol) stimulation of SR Ca2+-ATPase, or inhibition of Na+/K+-ATPase (ouabain). (2) In contrast, the blunted force–frequency relation can only slightly be improved with gingerol or low concentrations of isoproterenol and is further impaired with ouabain or high concentrations of isoproterenol. (3) Blocking SR Ca2+-ATPase with thapsigargin deteriorates post-rest and force–frequency behavior. (4) In human myocardial homogenates, gingerol stimulates SR Ca2+-uptake.

4.1 Reduced effectiveness of inotropic stimulation
β-adrenoceptor stimulation with isoproterenol exerts well-known positive inotropic effects in human myocardium [15,16] by increasing cAMP resulting in phosphorylation of cellular proteins including sarcolemmal Ca2+-channels, phospholamban and troponin I. By the first two mechanisms, isoproterenol increases myocardial Ca2+-turnover [17] and intracellular Ca2+-transients [18]. The reduced inotropic effect of isoproterenol in failing human myocardium has been attributed to a decreased number of β-adrenoceptors [19] and an increase in Gi-protein [20].

Gingerol is a cardiotonic agent from the rhizome of ginger and directly activates SR Ca2+-ATPase in mammalian myocardium [21,22], thereby increasing force probably without affecting phospholamban function. Gingerol did not affect function of sarcolemmal or myosin Ca2+-ATPase, actin-activated myosin ATPase and cAMP-phosphodiesterase activities [21]. Nevertheless, we cannot rule out the possibility that in human myocardium, gingerol may have additional, not yet identified effects. The present study shows for the first time a direct positive inotropic effect of gingerol in human myocardium. However, compared to guinea-pig atrial myocardium where [8]-gingerol increases twitch force by ~150%, the inotropic effect in failing human ventricular myocardium using [10]-gingerol is only ~15%. This difference may be due to differences in species (guinea-pig vs. human), type of myocardium (atrial vs. ventricular), inotropic agent ([8]- vs. [10]-gingerol) or application (addition to the organ bath vs. preincubation). Most importantly, decreased function and protein expression of SR Ca2+-ATPase in failing human myocardium [6] may contribute to this small inotropic effect of gingerol.

Ouabain exerts its positive inotropic effects by inhibiting Na+/K+-ATPase [23] with subsequent increases in intracellular Na+-concentration ([Na+]i) [24]. In consequence, Na+/Ca2+-exchange forward mode is inhibited, which limits Ca2+-extrusion during diastole. In addition, Na+/Ca2+-exchange reverse mode during systole might be activated, contributing to increased intracellular Ca2+-concentration ([Ca2+]i), SR Ca2+-load and twitch force. An increased sensitivity of cardiac glycosides in failing (vs. nonfailing) human myocardium had been described [25], and decreased activity [26] and protein expression of Na+/K+-ATPase in failing myocardium [27] was reported.

Thapsigargin, similarly as CPA [28] and 2,5-di(t-butyl)1,4-benzohydroquinone [29], inhibits SR Ca2+-ATPase and thereby Ca2+-reuptake. By this mechanism, thapsigargin decreases twitch force and prolongs twitch relaxation in rabbit [28] and human [30] myocardium similar to the present study.

4.2 Post-rest behavior
Potentiation of twitch force after a rest interval is an index for SR Ca2+-uptake capacity [1]. The SR plays a species-dependent role in excitation–contraction (E–C) coupling: in species with a strong SR like rat, rest potentiation of force can be found after rest intervals up to several minutes [1]. In contrast, in myocardium with a weak SR such as frog, post-rest potentiation is absent [31].

In nonfailing human myocardium, post-rest potentiation of force can be seen up to long rest intervals of several minutes (Fig. 1), with parallel increases in intracellular Ca2+-transients [2] and SR Ca2+-content [14]. However, in failing human myocardium there is post-rest decay of force at long rest intervals (Fig. 1) due to decreased Ca2+-transients [2] and SR Ca2+-content [14]. This altered post-rest behavior may be attributed to a reduced SR Ca2+-uptake capacity [5–8], due to reduced expression of SR Ca2+-ATPase on mRNA [32] and protein levels [6,33]. In addition, it was shown that protein expression [12] and function [10,11] of the Na+/Ca2+-exchanger are increased. Thus, less Ca2+ may be taken up back into the SR during rest and more Ca2+ may be removed from the cytosol by the Na+/Ca2+-exchanger resulting in rest-decay of force in failing myocardium.

Isoproterenol normalizes post-rest behavior in failing human myocardium possibly due to two mechanisms: increased Ca2+-influx during systolic activation and phosphorylation of phospholamban with enhanced SR Ca2+-uptake capacity. Especially, enhanced SR Ca2+-uptake capacity may prevent progressive unloading of the SR due to continuous leakage in the resting state and improved competition of SR Ca2+-ATPase vs. Na+/Ca2+-exchanger for cytosolic Ca2+. The present data indicate that defective SR Ca2+-accumulation is a major mechanism for altered post-rest behavior. However, our results are in contrast to findings of Davia and Harding [34] who described a decrease in post-rest force using isoproterenol in isolated myocytes from failing human hearts. This discrepancy may be due to different experimental conditions. Davia and Harding used a low basal stimulation frequency (0.2 Hz), low bath temperature (32°C), and [Ca2+]o of 1 mmol/l. Most importantly, they used only 0.01 µmol/l isoproterenol whereas in the present study 1 and 0.1 µmol/l were effective. Thus, reduced SR Ca2+-ATPase activity may not be stimulated sufficiently for net Ca2+ accumulation into the SR during rest at very low isoproterenol concentrations.

To investigate whether direct stimulation of SR Ca2+-ATPase can also normalize post-rest behavior we investigated the effects of gingerol [21,22]. Indeed, post-rest decay in failing human myocardium could be converted into a rest-dependent increase in force at long rest intervals. During rest, the SR seems to accumulate a larger amount of Ca2+ in the presence of gingerol, and less Ca2+ is available for extrusion by Na+/Ca2+-exchange. By this mechanism the physiological steady-state between SR Ca2+-ATPase and sarcolemmal Na+/Ca2+-exchanger is obviously restored, resulting in post-rest potentiation. Since gingerol does not alter myofilament Ca2+-sensitivity or other Ca2+-transport systems [21], it may be hypothesized that the normalized post-rest behavior depends on improved SR Ca2+-uptake which is impaired in failing myocardium.

In addition, ouabain normalizes post-rest behavior in failing myocardium. This may be due to decreased Ca2+-extrusion and/or increased Ca2+-influx via Na+/Ca2+-exchange reverse-mode and subsequent increases in [Ca2+]i and SR Ca2+-content [1]. Similar results could be found in isolated myocytes [34] from failing human hearts. Blocking Na+/Ca2+-exchange in rabbit myocardium converts the physiological rest decay of force into rest-potentiation probably by preventing cytosolic Ca2+-extrusion during rest due to relatively strong Na+/Ca2+-exchange, weak SR Ca2+-pump and low [Na+]i in rabbit [1,35].

To test whether post-rest behavior can further be deteriorated, we inhibited SR Ca2+-ATPase by thapsigargin [28]. In the presence of thapsigargin, post-rest potentiation was absent even at short, and was further deteriorated at long rest intervals. Similar results were obtained in isolated human myocardium using ryanodine [2]. These findings underline the importance for a functional SR for post-rest potentiation. Since diastolic tension did not increase in our experiments, the Na+/Ca2+-exchanger might extrude the greater fraction of Ca2+ that cannot be taken up by the SR during rest.

4.3 Force–frequency relation
Frequency-potentiation of contractile force is a potent inotropic mechanism in most mammalian hearts and in human myocardium [1,3,4], and is related to increased intracellular Ca2+-transients [5]. However, this positive force–frequency relation is blunted or even inversed in failing human myocardium (Fig. 1) associated with a parallel decline in Ca2+-transients [5]. Altered force–frequency behavior strongly correlates with reduced levels of SR Ca2+-ATPase and increased levels of Na+/Ca2+-exchanger in failing myocardium [6,9].

In the present study, stimulation of SR Ca2+-ATPase with low concentrations of isoproterenol slightly improved, whereas high concentrations further deteriorated force–frequency behavior in failing human myocardium. This is in agreement with previously published observations [15,16], but the underlying mechanism remains unclear. By further analyzing changes in diastolic tension we demonstrate, for the first time, that part of the benefit after low concentrations of isoproterenol results from improved diastolic function. The initial increase in force after isoproterenol was marginal at low, but substantial at higher concentrations. In contrast, relaxation parameters improved already at low concentrations of isoproterenol. However, it is known that β-adrenoceptor-stimulation with higher concentrations of isoproterenol overproportionally increases intracellular Ca2+-turnover [18]. Therefore, it may be speculated that the beneficial effect of low concentrations of isoproterenol on force-frequency behavior (which resulted in part from improved diastolic behavior) reflects improved Ca2+-ATPase capacity due to cAMP-dependent phosphorylation of phospholamban and reduced Ca2+-affinity for troponin C. While improved Ca2+-reuptake to the SR might normalize systolic and diastolic contractile function, reduced Ca2+-sensitivity of the myofilaments should primarily prevent diastolic dysfunction related to Ca2+-overload at high stimulation rates. In contrast, at intermediate and high concentrations of isoproterenol, the large increase in intracellular Ca2+-availability exhausts intracellular Ca2+-transport systems despite phospholamban phosphorylation, thereby further deteriorating force–frequency behavior. In addition, high concentrations of isoproterenol [16] or forskolin [36] convert a positive into a negative force–frequency relation even in nonfailing human myocardium.

In this study, gingerol increased SR 45Ca2+-uptake in myocardial homogenates from failing human hearts. Accordingly, gingerol increased Ca2+-accumulation in fragmented SR and 45Ca2+-uptake without changing 45Ca2+-efflux rate in isolated SR vesicles from dog hearts [21,22]. Functional experiments revealed that similar to low concentrations of isoproterenol, gingerol improved impaired force–frequency behavior in failing human myocardium. Since a close correlation between impaired force–frequency behavior and reduced expression of SR Ca2+-ATPase was described for failing human hearts [6], direct stimulation of Ca2+-ATPase by gingerol may result in partial normalization of SR Ca2+-uptake and hence, improved force–frequency behavior. Gingerol, unlike isoproterenol, does not increase [Ca2+]i by other mechanisms [21]. This further supports the hypothesis that reduced intracellular Ca2+-transients and force at higher stimulation rates predominantly result from reduced SR Ca2+-turnover, while potential changes in transsarcolemmal Ca2+-influx [37] may be of minor importance. Accordingly, we could recently demonstrate that SR Ca2+-content increases with higher stimulation rates in nonfailing, but fails to increase sufficiently in failing human myocardium [14]. However, probably due to diminished protein expression of Ca2+-ATPase, stimulation of SR Ca2+-uptake with gingerol only partially compensates reduced SR function in failing myocardium.

Ouabain deteriorated force–frequency relation in the present study mainly due to frequency-dependent increased diastolic tension. Similarly, Böhm et al. [15] showed a deterioration of the force–frequency relation using 0.02 µmol/l ouabain in failing human myocardium. Since it is also clinically appreciated that digitalis glycosides impair diastolic function [38], these compounds are not indicated in patients with hypertensive heart disease and prevailing diastolic abnormalities. In contrast to the present findings and previous studies, ouabain (0.01 µmol/l), in combination with the Na+-channel activator BDF 9148 (0.1 µmol/l), improved the force–frequency relation in failing myocardium [16]. The reason for the differences may be in part related to the fact that force in the present study increased by ~34% with ouabain, whereas Schwinger et al. did not report an increase in force with their low ouabain concentration. Furthermore, it has recently been demonstrated by our group that increased expression and activity of Na+/Ca2+-exchange in failing human myocardium is related to preserved diastolic function at high heart rates [9]. Therefore, Na+/K+-ATPase inhibition (and thus Na+/Ca2+-exchange inhibition) may clearly deteriorate force–frequency behavior partly related to diastolic dysfunction.

Thapsigargin has been shown to inhibit SR Ca2+-ATPase transport capacity [28]. Blockade of SR function allows to determine the role of SR Ca2+-ATPase on contractile function in failing myocardium. Thapsigargin reduced force and greatly prolonged relaxation time in this study. Upon increasing stimulation rate, the pathological force–frequency behavior was further impaired, partly due to rate-dependent increased diastolic tension. These effects may be brought about by delayed and reduced Ca2+-reuptake to the SR, with increasing functional consequences at high stimulation rates (which result in shorter diastolic periods for SR Ca2+-transport). In agreement, Davia et al. [30] found pronounced negative inotropic and lusitropic effects of thapsigargin in isolated myocytes from human hearts. Interestingly, in their study, the cardiodepressant action of thapsigargin was more pronounced in myocytes from nonfailing compared to failing hearts. This might indicate that contractile function depends to a greater extent on SR Ca2+-handling in normal as opposed to diseased human hearts. However, our results show that, though impaired, SR function still substantially contributes to contraction and relaxation in failing human myocardium.

4.4 Why is post-rest and force–frequency behavior distinctly affected
One interesting aspect of the present work is that post-rest and force–frequency behavior may be differentially affected by inotropic compounds. This holds especially true for those compounds that impair diastolic relaxation (ouabain, thapsigargin). In fact, post-rest contractions reflect SR Ca2+-load and -release, resulting from the relative contribution of SR Ca2+-ATPase and Na+/Ca2+-exchanger for cytosolic Ca2+-elimination [2]. Diastolic function is not a confounding factor under these conditions. This is obviously not the case for increasing stimulation rates, where diastolic dysfunction, possibly related to increased diastolic Ca2+ greatly contributes to altered active force generation. Therefore, post-rest contractions may more accurately reflect relative Ca2+-transport activities, but force–frequency behavior represents a more physiological intervention with clinical relevance.

4.5 Clinical relevance for heart failure therapy
The present data show that different inotropic interventions may very distinctly affect force–frequency behavior. Inhibition of Ca2+-elimination by ouabain impaired force–frequency behavior. Therefore, heart-rate dependent diastolic dysfunction and the need for vigorous heart rate control seems to be of particular importance in patients treated with cardiac glycosides. SR Ca2+-ATPase stimulation (at least to an extent that resulted in only small inotropic responses) improved force–frequency behavior. Therefore, stimulation of SR Ca2+-ATPase activity may be advantageous in patients with a negative force–frequency relation or important diastolic dysfunction. Gingerol is only used as a pharmacological tool. However, two concepts are currently under intensive investigation in our and other groups: (1) pyruvate, which improves hemodynamics in heart failure patients when applied intracoronary [39]. Pyruvate increases SR Ca2+-ATPase activity by increasing intracellular phosphorylation potential. (2) Gene therapy (e.g. adenovirus-mediated): overexpression of SERCA2a, which improves contractile function in phospholamban overexpressing rat cardiomyocytes [40].

Time for primary review 25 days.


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

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D. von Lewinski, E. Bisping, A. Elgner, J. Kockskamper, and B. Pieske
Mechanistic insight into the functional and toxic effects of Strophanthidin in the failing human myocardium
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