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Circulation. 1995;92:1169-1178

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(Circulation. 1995;92:1169-1178.)
© 1995 American Heart Association, Inc.

Articles

Alterations in Intracellular Calcium Handling Associated With the Inverse Force-Frequency Relation in Human Dilated Cardiomyopathy

Burkert Pieske, MD; Bodo Kretschmann, MS; Markus Meyer, MD; Christian Holubarsch, MD; Jörg Weirich, MD; Herbert Posival, MD; Kazatomo Minami, MD; Hanjörg Just, MD; Gerd Hasenfuss, MD

From the Medizinische Klinik III (B.P., B.K., M.M., C.H., H.J., G.H.) and Physiologisches Institut (J.W.), Universität Freiburg, and Klinik für Thorax- und Kardiovaskularchirurgie (H.P., K.M.), Herzzentrum Nordrhein-Westfalen, Bad Oeynhausen, Germany.

Correspondence to Burkert Pieske, MD, Medizinische Klinik III, Universität Freiburg, Hugstetter Str 55, 79106 Freiburg, Germany.


*    Abstract
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 *Abstract
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Background The present study was performed to test the hypothesis that the altered force-frequency relation in human failing dilated cardiomyopathy may be attributed to alterations in intracellular calcium handling.

Methods and Results The force-frequency relation was investigated in isometrically contracting ventricular muscle strip preparations from 5 nonfailing human hearts and 7 hearts with end-stage failing dilated cardiomyopathy. Intracellular calcium cycling was measured simultaneously by use of the bioluminescent photoprotein aequorin. Stimulation frequency was increased stepwise from 15 to 180 beats per minute (37°C). In nonfailing myocardium, twitch tension and aequorin light emission rose with increasing rates of stimulation. Maximum average twitch tension was reached at 150 min-1 and was increased to 212±34% (P<.05) of the value at 15 min-1. Aequorin light emission was lowest at 15 min-1 and was maximally increased at 180 min-1 to 218±39% (P<.01). In the failing myocardium, average isometric tension was maximum at 60 min-1 (106±7% of the basal value at 15 min-1, P=NS) and then decreased continuously to 62±9% of the basal value at 180 min-1 (P<.002). In the failing myocardium, aequorin light emission was highest at 15 min-1. At 180 min-1, it was decreased to 71±7% of the basal value (P<.01). Including both failing and nonfailing myocardium, there was a close correlation between the frequencies at which aequorin light emission and isometric tension were maximum (r=.92; n=19; P<.001). Action potential duration decreased similarly with increasing stimulation frequencies in nonfailing and end-stage failing myocardium. Sarcoplasmic reticulum 45Ca2+ uptake, measured in homogenates from the same hearts, was significantly reduced in failing myocardium (3.60±0.51 versus 1.94±0.18 (nmol/L) · min-1 · mg protein-1, P<.005).

Conclusions These data indicate that the altered force-frequency relation of the failing human myocardium results from disturbed excitation-contraction coupling with decreased calcium cycling at higher rates of stimulation.


Key Words: aequorin • excitation • contraction • sarcoplasmic reticulum • heart failure


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowResults
down arrowDiscussion
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Frequency potentiation of contractile force represents a potent inotropic mechanism in myocardium of most species, including nonfailing human myocardium.1 2 3 4 5 However, recent experiments in isolated human myocardium from end-stage failing hearts indicated that in the failing myocardium, frequency potentiation of contractile force is absent. Moreover, in most muscle strips from failing human hearts, the force-frequency relation was shown to be inverse.4 5 6 7 The relevance of these in vitro findings in the isolated human myocardium has been confirmed by clinical investigations: in patients with normal left ventricular function, hemodynamic parameters of myocardial contractility increased with increasing pacing rates during atrial or ventricular stimulation, whereas frequency potentiation of myocardial performance was absent in patients with heart failure.8 9

Frequency potentiation of contractile force was suggested to result from increased transsarcolemmal Ca2+ influx leading to greater filling of the sarcoplasmic reticulum (SR) and thus, a higher amount of Ca2+ available for release and activation of the contractile proteins.3 10 The subcellular defects underlying the inverse force-frequency relation in the failing human myocardium are unknown.

The altered force-frequency relation could result from a decreased calcium sensitivity or disturbed function of the contractile proteins at higher rates of stimulation despite normal calcium availability. Alternatively, intracellular calcium and thus, activator calcium to the contractile proteins may be reduced at higher frequencies. Myothermal measurements indicating that the total amount of calcium cycling is reduced significantly at a stimulation rate of 60 beats per minute (bpm) in the failing human myocardium support the latter possibility.11 Furthermore, the recent finding of a close correlation between force-frequency behavior of the human myocardium and SR Ca2+-ATPase protein levels may suggest that disturbed calcium cycling is a major underlying mechanism for the altered force-frequency relation of the failing human heart.12 However, none of the above-mentioned studies in human myocardium directly investigated the frequency-dependent changes of the intracellular Ca2+ transients under physiological experimental conditions.

Accordingly, the present study was performed to investigate the hypothesis that the positive force-frequency relation in nonfailing human myocardium results from increased free cytosolic calcium at higher rates of stimulation and that a frequency-dependent decline of systolic calcium transients is related to the inverse force-frequency relation in failing human hearts. The experiments were performed in isolated muscle strips from nonfailing human hearts and from hearts with end-stage failing dilated cardiomyopathy at 37°C and stimulation rates between 15 and 180 min-1 with the bioluminescent photoprotein aequorin. SR calcium uptake was measured in myocardium from the same hearts and related to frequency-dependent changes of the intracellular calcium signal. In addition, the influence of stimulation frequency on action potential duration was recorded.


*    Methods
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 *Methods
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Myocardial Tissue
Human left or right ventricular trabeculae were obtained from 6 nonfailing donor hearts that could not be transplanted for technical reasons and from 14 hearts with end-stage failing dilated cardiomyopathy at the time of heart transplantation. The mean age in the donor group was 39±7 years; three of the donors were women and three were men. No cardiac catheterization had been performed in the organ donor group, but none of the donors had a history of heart disease and all had normal left ventricular function. In the heart failure group, coronary artery disease or valvular disease had been excluded before transplantation. Clinical data of the patients are shown in Table 1Down.


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  		Table 1. Clinical Characteristics of the Patients

The study was reviewed and approved by the Ethical Committee of the University Clinics of Freiburg.

Muscle Strip Preparation
Immediately after explantation of the heart, the ventricles were carefully opened, and a thin layer of the subendocardial myocardium was excised for functional measurements. Special attention was given to avoiding damage of the fine trabecular network at the inner surface of the ventricle. In addition, transmural pieces of left ventricular myocardium from 3 of the 6 nonfailing hearts and from 7 of the 14 failing hearts were frozen in liquid nitrogen immediately after cardiectomy. This myocardium was stored at -80°C for later measurements of oxalate-facilitated SR 45Ca2+ uptake.

The excised myocardium for functional measurements was stored in a special cardioplegic solution oxygenated with carbogen (95% O2/5% CO2) and transported to the laboratory at room temperature. The solution contained (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 2,3-butanedione monoxime 30, plus insulin 10 IU/L. This cardioplegic solution was shown to protect the myocardium during transportation and from cutting injury at the time of muscle strip dissection.13 Its effects on the myocardium were shown to be fully reversible after washout.13 Small trabeculae were dissected with the help of a stereoscopic microscope. All preparation steps were carried out in the cardioprotective solution. The trabeculae were then mounted horizontally in a cylindrical glass cuvette between miniature clamps and connected to an isometric force transducer (OPT1L, Scientific Instruments) and superfused with a carbogen-bubbled modified Krebs-Henseleit solution of the composition given above, with the exception that 2,3-butanedione monoxime was omitted. Punctate stimulation via a platinum electrode at the end of the muscle was used. After the muscle strips were initially prestretched with a force of 1 mN, they were allowed to equilibrate for 30 to 60 minutes at a stimulation frequency of 1 Hz and stimulation voltage 20% above threshold. Thereafter, the muscles were gradually stretched along their length-tension curve in 0.05-mm steps until maximum isometric tension was reached.

Aequorin Measurements
By the time of complete mechanical stabilization of the trabecular strips at maximum isometric tension, electrical stimulation was switched off for 5 minutes, and the Ca2+-regulated bioluminescent photoprotein aequorin was macroinjected into the quiescent muscle just beneath the endocardium.14 The aequorin light signal was detected with a photomultiplier tube (XP 2802, Philipps) vertically mounted with its cathode just above the glass cuvette containing the aequorin-loaded muscle. To increase the optical efficiency of the system, an ellipsoidal mirror was placed beneath the glass cuvette, reflecting photons to the photomultiplier tube. Light emissions (in millivolts of amplifier output) and force signals were recorded simultaneously on-line on a personal computer and an oscilloscope with signal-averaging function (Nicolet PRO 10C, Nicolet Instrument Corp) as well as on a strip-chart recorder (WR 3310, Graphtec) for original registration. Fifty light transients were averaged at each experimental step to increase the signal-to-noise ratio. The experimental protocol was started at the time when the aequorin light signals were completely stable.

Aequorin was purchased in lyophilized form from Prof John R. Blinks at the Friday Harbor Laboratories. Lyophilized aequorin (1 mg) was dissolved in 700 µL quartz-distilled water to minimize Ca2+ contamination.

Experimental Protocol
After stable light and force signals at 60 min-1 had been obtained for at least 10 minutes, stimulation frequency was reduced to 15 min-1. From this frequency, the stimulation rate was increased stepwise up to a maximum frequency of 180 min-1. Isometric twitch myograms and aequorin light signals were sampled during steady-state conditions at the following stimulation frequencies: 15, 30, 60, 90, 120, 150, and 180 min-1. Twitch tension is defined as the active tension developed during the isometric twitch. It is the amplitude of the twitch signal between peak systolic tension and diastolic tension at the end of the stimulus interval. Accordingly, aequorin light emission is defined as the amplitude of the aequorin light signal between the peak systolic light emission and the diastolic baseline value. The amplitude of the aequorin light signal (millivolts of amplifier output) was compared with the amplitude of the isometric twitch tension (mN/mm2) at each stimulation frequency. In addition to amplitudes of twitch and aequorin light signal, total twitch time, time to 50% relaxation, and time to 95% relaxation of the isometric twitch and time to 50% decline and time to 80% decline of the aequorin light signal were evaluated (see Table 2Down). In one set of experiments, the force-frequency relation was investigated in muscle strip preparations from 3 nonfailing hearts after preincubation for 30 minutes with the ß-adrenergic receptor blocker propranolol (1 µmol/L). Cross-sectional area of the muscle for normalization of force values was determined as the ratio of blotted muscle weight to muscle length. Average cross-sectional area of the muscle strips was 0.40±0.04 mm2 (0.15 to 0.66 mm2). Experiments were performed in 7 muscle strips from 5 nonfailing hearts (2 from the right, 5 from the left ventricle) and in 12 muscle strips from 9 end-stage failing hearts (5 from the right, 7 from the left ventricle).


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  		Table 2. Relaxation Parameters of Isometric Twitches and Aequorin Light Signals

SR 45Ca2+ Uptake Assay
Oxalate-facilitated Ca2+ uptake was measured immediately after homogenization with the Millipore filtration system (Millipore Corp). The assay was performed according to the method described by Pagani and Solaro.15 For measurement of calcium uptake, small samples of the frozen myocardium were minced with scissors and knife on a ice-cold rubber surface. During this procedure, care was taken to remove fat, vessels, and epicardium. Homogenization was performed in 10 mL of ice-cold solution containing 25 mmol/L imidazole (pH 7.0) for four 10-second periods in a Polytron homogenizer (PT-K, Kinematica). An aliquot of the homogenate was transferred into the uptake medium containing (in mmol/L, final concentration) KCl 100, imidazole 40, potassium oxalate 5, MgCl2 4.5, NaCl2 10, sodium ATP 2.5, creatine phosphate 3, and 2 IU/mL creatine phosphokinase (pH 7.0). After an equilibration time of 5 minutes (37°C), the assay was started by addition of CaCl2 (25 µmol/L, 0.185 µCi 45Ca2+/mL) and EGTA (15.5 µmol/L). Aliquots of the reaction medium (100 µL) were taken after 30, 60, 90, and 120 seconds, filtered through a 0.45-µm Millipore filter, and rapidly washed with ice-cold 0.6 mol/L KCl, 5 mmol/L NaN3, and 20 mmol/L imidazole to stop the uptake. Radioactivity was determined by liquid scintillation spectroscopy. The calcium uptake rate was calculated from the linear regression analysis of the four time points from the slope relating calcium uptake to time. Each uptake rate used in the calculations is the mean of three uptake measurements. Free calcium concentrations were calculated by a computer program and expressed as nmol/L Ca2+ · min-1 · mg protein-1. Protein was determined by the method described by Bradford.16

Action Potential Measurements
In a further set of experiments, the influence of stimulation frequency on action potential characteristics was investigated in 7 muscle strip preparations from 5 end-stage failing hearts (4 from the left and 3 from the right ventricle) and in 1 left ventricular muscle strip preparation from a nonfailing human heart. The muscle strip preparations were placed in an organ bath and superfused with a carbogen-bubbled modified Krebs-Henseleit solution of the above composition at 37°C. The preparations were electrically stimulated via a punctate electrode with rectangular pulses of 0.2-ms duration, voltage 20% above threshold. Transmembrane action potentials were recorded by means of conventional 3 mol/L KCl–filled glass microelectrodes (resistance, 5 to 11 M{Omega}). Action potentials were displayed on a digital storage oscilloscope (Nicolet 2090) and stored on a 486 personal computer for data analysis. Only experiments in which a single impalement could be maintained throughout control and experimental periods were evaluated. After impalement, stimulation frequency was increased stepwise from 15 to 180 bpm, and action potentials were recorded at each stimulation frequency after complete stabilization of the signals. Simultaneously, frequency-dependent changes in isometric force were evaluated in muscle strip preparations from the same hearts according to the protocol described above.

Statistical Analysis
Average values are given as mean±SEM. Comparison within one group of myocardium was performed with the paired t test. If multiple values within one group were compared, the paired t test followed by the Bonferroni-Holm equation17 was used. Comparison between different groups of myocardium was performed with the unpaired t test. Correlations were examined by linear regression analysis. Differences were considered significant at P<.05.


*    Results
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 up arrowIntroduction
 up arrowMethods
*Results
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In the nonfailing and failing human myocardium, isometric twitch tension and aequorin light emission critically depended on stimulation frequency. Fig 1Down shows original recordings of experiments in an aequorin-loaded muscle strip from a nonfailing heart (top) and a failing heart (bottom). In both muscle preparations, stimulation frequency was increased from 30 to 120 min-1 and then reduced to 30 min-1 again. When stimulation frequency was increased from 30 to 120 min-1, the nonfailing myocardium responded with a pronounced increase in the amplitude of the light signal. The increase in light emission was accompanied by an increase in twitch tension amplitude with only minimal changes in diastolic tension. In the failing myocardium, the increase in stimulation rate from 30 to 120 min-1 resulted in a pronounced decrease in the amplitude of the light signal. The change in light amplitude was accompanied by a pronounced decrease in the amplitude of the isometric twitch, with only a slight increase in diastolic tension. The complete reversibility in light and tension changes in both preparations were shown by reducing the stimulation rate back to 30 min-1.



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  		Figure 1. Original recordings of aequorin light and isometric tension signals from aequorin-loaded left ventricular trabecular strips from a nonfailing heart (top) and a heart with end-stage failing dilated cardiomyopathy (DCM) (bottom). Stimulation frequency was increased from 30 to 120 min-1. After steady-state conditions were reached, stimulation frequency was reduced back to 30 min-1. Upper tracing in each panel, aequorin light signal. Lower tracing in each panel, isometric twitch tension.

The differences between nonfailing and failing myocardium with respect to isometric force development and aequorin light emission could be seen over the whole frequency range. Fig 2Down shows typical experiments in a muscle strip from a nonfailing and from an end-stage failing heart. The change in force or aequorin light emission is given in absolute values. As becomes evident, the isometric twitch tension of the preparation from the nonfailing heart increased from 7.6 mN/mm2 at 15 min-1 to 20.9 mN/mm2 at 150 min-1. This increase in force was associated with a parallel increase in aequorin light emission from 380 to 776 mV. In the muscle strip from the failing heart, isometric twitch tension was maximal at 30 min-1 (6.4 mN/mm2) and then declined to 4.1 mN/mm2 at 180 min-1. This decline in force was associated with a parallel decline in the aequorin light signal from 398 mV at 15 min-1 to 198 mV at 180 min-1.



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  		Figure 2. Graph showing influence of stimulation frequency on intracellular Ca2+ transients and isometric twitch tension in a muscle strip from a nonfailing heart and a heart with end-stage failing dilated cardiomyopathy (DCM). Changes in the Ca2+ transients, as reflected by the aequorin light emission (triangles), are given in millivolts of amplifier output (right ordinate); changes in the isometric twitch tension (circles) are given in mN/mm2 (left ordinate).

The average values for force and aequorin light signals in nonfailing myocardium are shown in Fig 3Down. In all experiments, twitch tension and aequorin light emission were lowest at 15 min-1 and increased considerably with higher stimulation rates. Maximum twitch tension and aequorin light emission were reached at stimulation frequencies between 120 and 180 min-1. Maximum average twitch tension was reached at a frequency of 150 min-1 and was increased to 212±34% of the basal value at 15 min-1 (P<.05). At 180 min-1, tension was still increased to 185±29% of the basal value (P<.05). The frequency dependence of the average aequorin light signal parallels that of the twitch tension. Light emission was lowest at 15 min-1 and reached its average maximum at a stimulation frequency of 180 min-1, when it was increased to 218±39% of the basal value at 15 min-1 (P<.01). Frequency-dependent changes of relaxation parameters of aequorin light signals and isometric twitches are given in Table 2Up.



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  		Figure 3. Graph showing influence of stimulation frequency on aequorin light emission and isometric twitch tension in nonfailing human myocardium. Values are given as percent change from the basal value at 15 min-1. Each point represents the mean±SEM from 7 preparations from 5 hearts. Basal twitch tension was 6.3±1.7 mN/mm2 at 15 min-1; cross-sectional area was 0.32±0.04 mm2.

To evaluate the possibility that the positive force-frequency relation in nonfailing myocardium might be due to increased catecholamine release during higher stimulation rates, experiments were performed after ß-adrenergic receptor blockade with propranolol. Under the influence of 1 µmol/L propranolol, the positive force-frequency relation was similar. Maximum tension was reached at 150 min-1 and was increased to 256±18% of the basal value at 15 min-1 (n=3). This indicates that catecholamine release and subsequent ß-adrenergic receptor stimulation is not the cause of the positive force-frequency relation in nonfailing myocardium.

The average values for force and aequorin light signals in the failing myocardium are shown in Fig 4Down. In isolated myocardium from hearts with end-stage failing dilated cardiomyopathy, isometric twitch tension and aequorin light signals tended to decrease with higher stimulation rates. Although there was some variation in the optimal stimulation frequency for each individual muscle strip (15 to 90 min-1), average isometric twitch tension was maximum at a stimulation frequency of 60 min-1, where it attained 106±7% of the basal value at 15 min-1 (P=NS). At stimulation rates >60 min-1, force of contraction declined continuously in most muscle strip preparations. At 180 min-1, twitch tension was reduced to 62±9% of the basal value at 15 min-1 (P<.002). As is also evident from Fig 4Down, the average aequorin light signal decreased with higher rates of stimulation. The decline of the aequorin light emission parallels that of the isometric force. At 180 min-1, the average aequorin light signal was decreased to 71±7% of the value at 15 min-1 (P<.01). Frequency-dependent changes of relaxation parameters of aequorin light signals and isometric twitches are given in Table 2Up.



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  		Figure 4. Graph showing influence of stimulation frequency on aequorin light emission and isometric twitch tension in end-stage failing human myocardium. Values are given as percent change from the basal value at 15 min-1. Each point represents the mean±SEM from 12 preparations from 9 hearts. Basal twitch tension was 8.4±1.5 mN/mm2; cross-sectional area was 0.47±0.03 mm2.

Including nonfailing (n=7) and failing (n=12) myocardium, there was a significant correlation (r=.92; P<.001) between the frequencies at which peak aequorin light signal and peak isometric twitch tension were reached (Fig 5Down). The correlation between the two parameters was also statistically significant when the analysis was performed in myocardium from hearts with dilated cardiomyopathy exclusively (r=.88; P<.001). These correlations demonstrate that parallel changes in light frequency and force frequency occur in the human myocardium.



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  		Figure 5. Graph showing the relation between the stimulation frequencies at which the aequorin light signals and the isometric twitch tension signals were maximum (Fmax). Each data point reflects one experiment. {circ}, End-stage failing myocardium; {bullet}, nonfailing myocardium.

Oxalate-facilitated 45Ca2+ uptake by the SR was measured in homogenates from 3 of the 5 nonfailing and 7 of the 9 failing hearts. In the nonfailing myocardium, 45Ca2+ uptake was 3.60±0.51 (nmol/L) · min-1 · mg protein-1. However, in the failing myocardium, 45Ca2+ uptake was reduced significantly, to 1.94±0.18 (nmol/L) · min-1 · mg protein-1 (P<.05 versus nonfailing; Table 3Down). There was a clear separation between nonfailing and failing myocardium with respect to Ca2+ uptake and the stimulation frequency when aequorin light emission (ie, intracellular Ca2+ transient) was maximal: In the nonfailing myocardium, Ca2+ uptake was high and maximal aequorin light emission was reached at a high stimulation rate. In end-stage failing myocardium, Ca2+ uptake was low and maximal aequorin light emission was reached at a low stimulation rate (Fig 6Down).


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  		Table 3. Relation Between SR 45Ca2+ Uptake and the Stimulation Frequency at Which Aequorin Light Emission Was Maximal



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  		Figure 6. Graph showing relation between the stimulation frequency at which aequorin light emission was maximal (Lmax) (in beats per minute) and the sarcoplasmic reticulum 45Ca2+ uptake capacity [(nmol/L) · min-1 · mg protein-1] from homogenates of the same hearts. Data points reflect average values from each heart. {circ}, End-stage failing myocardium; {bullet}, nonfailing myocardium.

Frequency-dependent changes in isometric force may be related to changes in action potential duration resulting from changes in transsarcolemmal Ca2+ flux. Therefore, frequency-dependent changes in action potential duration (time to 50% repolarization and time to 90% repolarization, APD50 and APD90) were measured in muscle strip preparations from 5 end-stage failing hearts and 1 nonfailing heart. Simultaneously, the force-frequency relation was characterized in muscle strip preparations from the same hearts. Fig 7Down shows typical superimposed recordings of action potentials at 30 and 120 bpm and the corresponding isometric twitches from an end-stage failing and a nonfailing heart. The action potentials were characterized by a rapid upstroke followed by a gradually increasing speed of repolarization without a marked point of termination of the plateau phase. Such action potentials are typical for ventricular myocardium of mammals18 and humans.19 In both types of myocardium, action potential duration decreased with the higher stimulation frequency, whereas isometric twitch tension increased in the nonfailing myocardium but decreased in the end-stage failing myocardium. The average values for all stimulation frequencies are shown in Fig 8Down. It is obvious that in both types of myocardium, APD50 and APD90 shorten with increasing stimulation frequency. In the nonfailing myocardium, the frequency-dependent decline in action potential duration is associated with an increase in isometric twitch tension. In the end-stage failing myocardium, isometric twitch tension tends to decline with higher stimulation frequencies and shorter action potential duration. To evaluate whether the force-frequency behavior of the individual hearts may be related to frequency-dependent changes in action potential configuration, the change in action potential duration (APD50, APD90) after an increase in stimulation frequency from 30 to 120 min-1 was correlated with the parallel frequency-dependent change in twitch tension. In dilated cardiomyopathy, no significant correlation between the frequency-dependent change in action potential duration and force was observed (for APD50, r=.6377, P<.173; for APD90, r=.5218, P<.288; n=6).



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  		Figure 7. Superimposed action potential recordings (upper tracing) and isometric twitches (lower tracings) from muscle strip preparations from a nonfailing heart and from an end-stage failing heart. Recordings were performed at a stimulation frequency of 30 beats per minute (bpm) and after increasing stimulation frequency to 120 bpm. Membrane potential just before the upstroke was -90.0 and -92.1 mV at 30 and 120 bpm, respectively, in the muscle from the nonfailing heart and -91.8 and -93.1 mV at 30 and 120 bpm, respectively, in the muscle from the end-stage failing heart. Mean values in the heart failure group (n=7) were -87.8±2.5 mV at 30 bpm and -88.8±2.2 mV at 120 bpm.



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  		Figure 8. Graphs showing stimulation-frequency–dependent changes of action potential duration to 50% and 90% repolarization (APD50 and APD90; top) and isometric twitch tension (bottom) in human nonfailing and end-stage failing myocardium. One muscle strip preparation from 1 nonfailing heart and 7 muscle strip preparations from 5 end-stage failing hearts.


*    Discussion
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up arrowResults
*Discussion
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Frequency potentiation of contractile force is considered to be a major physiological mechanism for the regulation of myocardial performance.1 3 It is now well established that frequency potentiation of contractile force occurs in the nonfailing human heart,4 5 but substantial alterations of the force-frequency relation have been observed in the failing human heart under in vitro as well as in vivo conditions.4 5 6 7 8 9

Mulieri et al4 were the first to demonstrate the blunted force-frequency relation in human end-stage failing myocardium under physiological conditions. These authors speculated that a reduced Ca2+ activation of the contractile proteins at higher rates of stimulation may be the underlying defect. However, simultaneous measurements of intracellular Ca2+ transients and isometric force have never been performed in human myocardium under physiological conditions.

Gwathmey et al7 and Morgan,20 using the photoprotein aequorin to measure intracellular Ca2+ concentrations, did not observe stimulation-interval–related differences in the peak systolic Ca2+ transients between nonfailing and end-stage failing human myocardium. However, these experiments were performed at an unphysiological low temperature (30°C) and at low stimulation frequencies (0.6 to 60 bpm). It is important to note that frequency-dependent changes of contractile force depend critically on experimental temperature and the frequency range investigated.13 21

Since simultaneous force and Ca2+ measurements have never been performed under physiological conditions as to temperature and stimulation rate, it was the purpose of the present study to investigate frequency dependence of contractile force and intracellular Ca2+ transients in nonfailing and end-stage failing human myocardium at a physiological temperature (37°C) and over the whole physiological frequency range. We tested the hypothesis that the inverse force-frequency relation in the failing human myocardium is associated with reduced aequorin light emission, and thus, reduced intracellular Ca2+ transients, at higher rates of stimulation.

The present data confirm that the force-frequency relation is positive in nonfailing human myocardium and that it is flattened or inverse in failing myocardium. Furthermore, the present aequorin measurements strongly indicate that the positive force-frequency relation in the nonfailing myocardium results from frequency-dependent increases in the intracellular Ca2+ transients and that the altered force-frequency relation in the failing myocardium is attributable to disturbed calcium cycling processes in the diseased human heart. This can be seen from the parallel changes of isometric force and aequorin light signals in the different types of myocardium. Changes in the amplitude of the aequorin light signal reflect changes in the free intracellular Ca2+ concentration relevant for activation of contractile proteins.22 In nonfailing myocardium, higher stimulation rates result in an increase in the amplitude of the aequorin light signal, indicating an increase in the intracellular free Ca2+ concentrations and a parallel rise in twitch tension. In contrast, the decline in isometric tension at higher rates of stimulation in the failing myocardium is associated with a decline in the amplitude of the aequorin light signals, indicating a decrease in the free intracellular Ca2+ concentration. Furthermore, the close correlation between the frequencies at which maximum isometric force and light emission are reached strongly indicates that the frequency dependence of the intracellular free calcium concentration determines the force-frequency relation in the human myocardium. On the other hand, these findings make it unlikely that changes in calcium sensitivity or changes in the behavior of the contractile proteins are a major cause of the altered force-frequency relation in the failing human myocardium.

The present findings are in contrast to data published by Gwathmey et al7 and Morgan.20 These investigators observed an inverse force-frequency relation but a positive aequorin light-frequency relation in failing compared with nonfailing human myocardium (after stimulation frequency was increased from 20 to 60 bpm). From this dissociation between force and light after an increase in stimulation frequency, the authors concluded that abnormalities of contractile function in failing human myocardium are not due to decreased availability of intracellular Ca2+ but more likely reflect differences of myofibrillar Ca2+ responsiveness. However, the decline in force was due primarily to an increase in diastolic tone (contracture; Reference 7, Fig 11). The decrease in force amplitude due to contracture occurred despite an increase in peak amplitude of the Ca2+ transient when stimulation frequency was raised from 20 to 60 bpm (Reference 7, Table 1Up). We believe that the different results between our work and that of Gwathmey et al are related to differences in experimental conditions. In the studies by Gwathmey et al, the force-frequency relation was studied in the range from 20 to 60 bpm. A positive force-frequency relation (this study; see also Mulieri et al4 ) or aequorin light-frequency relations in failing human myocardium in the low stimulation frequency range up to 60 bpm is not an uncommon finding. In fact, in our study, in 5 of 11 muscle strip preparations from failing hearts, aequorin light emission was maximal at 60 bpm and declined only at higher stimulation frequencies. Therefore, since measurements were performed only at low stimulation frequencies in the study by Gwathmey et al, the negative portion of the aequorin light-frequency range may have been above the frequency range investigated. In addition, lowering the temperature from 37°C to 30°C results in a considerable prolongation of the isometric twitch due to a reduced rate of Ca2+ elimination from the cytosol and alterations of cross-bridge behavior. Accordingly, as shown by Gwathmey et al, a stimulation rate of 60 bpm results in incomplete relaxation and thus, a rise in diastolic tension. Alternatively, acidosis or phosphate accumulation in the core of the muscle strip preparations might have been the cause of the dissociation between tension development and intracellular Ca2+ transients.

In this study, only minor changes in diastolic tension and diastolic aequorin light emission were observed at higher stimulation rates. This is in good agreement with the data of Mulieri et al.23 Of note, because of its binding characteristics, the Ca2+ indicator aequorin is very sensitive to changes in peak systolic Ca2+ concentrations but less sensitive in the low Ca2+ concentration range during diastole.22 Therefore, minor changes in diastolic Ca2+ concentration may not be reliably detected by use of the photoprotein aequorin.

In the present study, we did not observe two distinct components of the aequorin light signal (L1 and L2) in dilated cardiomyopathy. This is in contrast to Gwathmey et al,24 who demonstrated two distinct light components in failing human myocardium. The distinct light components were most pronounced in hypertrophic obstructive cardiomyopathy but were also detected in dilated cardiomyopathy. However, Beuckelmann et al,25 using the fluorescent Ca2+ indicator fura-2, did not observe distinct light components in isolated myocytes from hearts with end-stage failing dilated cardiomyopathy at 35°C. Furthermore, a contractile counterpart to L2 has never been shown. Therefore, we believe that the second component of the aequorin light signal may be present primarily at low temperature.

Disturbed calcium cycling as the cause of reduced tension development in the failing human myocardium is consistent with recent myothermal measurements showing that the total amount of calcium cycling is reduced at physiological stimulation rates in the failing human heart.11 Furthermore, reduced peak systolic calcium concentrations were suggested from fura-2 measurements in isolated myocytes from failing human myocardium.25

The question arises as to which subcellular alterations may be the underlying cause for the disturbed Ca2+ cycling in the failing human myocardium. The positive force-frequency relation observed in most types of mammals and in nonfailing human myocardium may result from an increased amount of Ca2+ released from the SR at higher rates of stimulation.3 10 Increased Ca2+ release is believed to be the consequence of an increased loading of the SR with Ca2+, which in turn results from a larger amount of Ca2+ entering the myocytes per unit of time at higher stimulation frequencies.3

Accordingly, the present data indicate that the altered force-frequency behavior in the failing human myocardium may result from decreased instead of increased SR Ca2+ release at higher stimulation frequencies. This is consistent with data from Orchard and Lakatta,26 who suggested that the negative force-frequency relation in the rat is due to diminished SR Ca2+ release. Diminished SR Ca2+ release could result from (1) a reduced amount of trigger calcium entering the cell through the L-type calcium channels, (2) a defect of the SR Ca2+ release channel, or (3) a decreased amount of Ca2+ available at the SR Ca2+ release sites. The latter could result from disturbed Ca2+ reuptake into the SR by the SR Ca2+ pumps and therefore SR Ca2+ depletion or from increased transsarcolemmal Ca2+ extrusion by the Na+/Ca2+ exchanger or the sarcolemmal Ca2+ ATPase.

The action potential in heart muscle generally shows a plateau phase at positive potential, which has been attributed to the inward Ca2+ current, ICa.27 Furthermore, it was shown that the relation between action potential and force yields information about Ca2+-related currents.28 29 In our study, action potential duration decreased with higher stimulation frequencies in both types of myocardium. This is consistent with data from Schouten et al,30 who found a 27% decrease in APD20 after increasing stimulation frequency from 0.1 to 1.0 Hz in isolated human ventricular myocardium. However, although action potential duration decreased with higher stimulation rates in both nonfailing and failing myocardium, isometric twitch tension increased in nonfailing and declined in failing myocardium at higher stimulation rates. In addition, no significant correlation between action potential duration and the frequency-dependent change in isometric twitch tension could be obtained in end-stage failing myocardium. There was a tendency for action potential duration to be longer in failing than in nonfailing myocardium at stimulation rates <120 bpm. This is in agreement with previously published reports in human isolated cardiomyocytes31 or muscle strip preparations.7 Therefore, since the duration of the plateau phase of the action potential is considered to be an index for transsarcolemmal Ca2+ influx, it is unlikely that the inverse force-frequency relation is due to alterations of transsarcolemmal Ca2+ influx. Furthermore, Beuckelmann et al32 did not find altered Ca2+ currents in isolated myocytes from failing human myocardium due to dilated cardiomyopathy.

The question arises as to whether a disturbed Ca2+ release from the SR may contribute to the finding of the inverse force-frequency relation in the failing myocardium. A decrease in the Ca2+ transient could originate from a reduced Ca2+ release via the SR Ca2+ release channels (ryanodine receptor) despite a normal amount of Ca2+ stored within the SR or an increased Ca2+ leak from the SR that prevents normal Ca2+ accumulation. Few experimental data regarding these two possibilities are actually available for human myocardium. When human nonfailing and human end-stage failing dilated cardiomyopathies are directly compared, no significant alteration in mRNA expression of the ryanodine receptor has been observed.33 Arai et al,34 however, found a significant inverse relation between mRNA expression of atrial natriuretic factor and Ca2+ release channels in failing dilated cardiomyopathy. Holmberg and Williams35 did not find abnormal functional activity of single SR Ca2+ release channels under voltage-clamp conditions from end-stage failing human hearts. However, the latter authors compared their data with the activity of SR Ca2+ release channels from normal sheep and canine myocardium, which may not completely reflect the situation in the normal human heart. D'Agnolo et al36 found an increased threshold for caffeine to release Ca2+ from the SR in idiopathic dilated cardiomyopathy and postulated an abnormal gating mechanism of the Ca2+ release channel in this disease state. With respect to an increased leakage of Ca2+ from the SR, some disease states such as malignant hyperthermia have been related to pathological Ca2+ efflux through SR Ca2+ release channels in human37 and porcine38 skeletal muscle. However, similar findings have not been described for the human heart. Therefore, at the present time, we cannot exclude the possibility that alterations at the level of the SR Ca2+ release channels may contribute to the pathological force-frequency behavior in human dilated cardiomyopathy.

There is increasing evidence in support of the hypothesis that diminished Ca2+ reuptake into the SR may be of major relevance for contractile dysfunction in the failing human heart. It was shown recently that the expression of the Ca2+-ATPase of the SR is reduced at the level of the mRNA as well as the protein in the failing human myocardium.12 34 39 40 41 Furthermore, a significant correlation between the protein levels of the SR Ca2+-ATPase and the force-frequency behavior of the failing human myocardium has now been demonstrated.12 These data, in conjunction with the present findings, provide considerable evidence that the reduced tension development at higher frequencies in the failing myocardium may be due to decreased SR Ca2+ release resulting from SR Ca2+ depletion. The latter may occur because at higher stimulation rates, the time available for Ca2+ transport into the SR per cardiac cycle shortens, which, in the presence of a decreased number of SR calcium pumps, may cause insufficient Ca2+ reuptake.

This hypothesis is further supported by our finding that in the failing human myocardium, Ca2+ uptake capacity to the SR is significantly reduced compared with nonfailing myocardium. A low Ca2+ uptake capacity was associated with a blunted frequency potentiation of contractile force. A reduced Ca2+ uptake capacity in failing human myocardium was first described by Limas et al,42 but Movsesian et al43 did not observe a reduced SR Ca2+ uptake capacity in human dilated cardiomyopathy. The latter authors, however, performed their uptake measurements in highly purified SR vesicles, which may attenuate the effect of a decreased expression of SR Ca2+ pumps on SR Ca2+ uptake.

Under conditions of decreased SR calcium accumulation, one would expect diastolic calcium to increase and cause diastolic activation of the contractile proteins. Since in the present study, as well as in previous investigations,4 23 no major changes in diastolic tension were observed, alternative routes for calcium elimination from the myoplasm must exist. Intracellular free Ca2+ may be bound by Ca2+ sinks within the cells, such as troponin C, mitochondria, or other Ca2+-binding proteins.44 45 46 Alternatively, Ca2+ may be eliminated across the sarcolemma to the extracellular space by Ca2+ transport systems, such as the sarcolemmal Ca2+-ATPase or the Na+/Ca2+ exchanger.47 48 Compensatory Ca2+ elimination by the Na+/Ca2+ exchanger may be likely, because it was shown recently that the expression of this protein is increased in failing human hearts concomitantly with a decreased expression of SR Ca2+-ATPase.41 Furthermore, a reduced Ca2+ uptake capacity of the SR would favor extrusion of cytosolic Ca2+ to the extracellular space via Na+/Ca2+ exchange and possibly sarcolemmal Ca2+-ATPase. This could result in a net loss of Ca2+ from the SR and thus, from the cell. Therefore, in the failing myocardium, both a decrease in SR Ca2+ uptake capacity and an increase in Na+/Ca2+ exchange activity may contribute to the decline in systolic tension generation and the amplitude of the aequorin light signal at higher stimulation rates.

In summary, the present study shows that frequency dependence of isometric force generation is closely related to the free intracellular Ca2+ concentrations in the human myocardium. In nonfailing myocardium, increasing stimulation frequencies lead to an increase in the intracellular Ca2+ transients and thus, to an increase in systolic force generation. In the failing myocardium, however, in which substantial alterations occur with respect to proteins involved in intracellular Ca2+ handling, increasing stimulation frequencies lead to a decrease in intracellular Ca2+ transients and thus, to a decrease in systolic force generation. Since SR Ca2+ uptake capacity was reduced in these hearts, this defect may contribute to the observed alterations in excitation-contraction coupling. However, we cannot exclude the possibility that decreased transsarcolemmal Ca2+ influx, increased Ca2+ elimination by the Na+/Ca2+ exchanger, or defects on the level of the Ca2+ release channel of the SR contribute to the alterations in intracellular Ca2+ handling underlying the inverse force-frequency relation in human dilated cardiomyopathy.


*    Acknowledgments
 
This study was supported by Deutsche Forschungsgemeinschaft grant HA 1233/3-1. The authors wish to thank Jim Morgan and Jianxun Wang, Beth Israel Hospital, Harvard University, Boston, Mass, as well as John Blinks, University of Washington, Friday Harbor, for their support in establishing the aequorin technology in isolated heart muscle.

Received September 29, 1994; revision received February 14, 1995; accepted February 27, 1995.


*    References
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