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Cardiovascular Research 1998 37(2):478-488; doi:10.1016/S0008-6363(97)00280-0
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Frequency dependence of Ca2+ release from the sarcoplasmic reticulum in human ventricular myocytes from end-stage heart failure

Karin R Sipidoa,*, Tania Stankovicovaa, Willem Flamengb, Johan Vanhaeckea and Fons Verdonckc

aLaboratory of Experimental Cardiology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
bCenter for Experimental Surgery and Anesthesiology, University of Leuven, Leuven, Belgium
cInterdisciplinary Research Center, University of Leuven-Kortrijk, Leuven, Belgium

* Corresponding author. Tel. (+32-16) 34 71 53; Fax (+32-16) 34 58 44; E-mail: karin.sipido@med.kuleuven.ac.be

Received 22 July 1997; accepted 17 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objectives: Human cardiac muscle from failing heart shows a decrease in active tension development and a rise in diastolic tension at stimulation frequencies above 50–60 beats/min due to both systolic and diastolic dysfunction. We have investigated underlying changes in cellular [Ca2+]i regulation. Methods: Single ventricular myocytes were isolated enzymatically from the explanted hearts of transplant recipients with ischemic cardiomyopathy (nhearts=5, ncells=15) or dilated cardiomyopathy (nhearts=6, ncells=19). Cells were studied during whole-cell patch clamp with fluo-3 and fura-red as [Ca2+]i indicators (36±1°C). Results: In current clamp mode (action potential recording), the amplitude of Ca2+ release from the sarcoplasmic reticulum (SR) decreased at stimulation frequencies above 0.5 Hz; this decrease was more pronounced for cells from dilated cardiomyopathy. Diastolic [Ca2+]i increased at 1 and 2 Hz for both groups. Action potential duration (APD90) decreased with frequency in all cells; in addition there was a drop in plateau potential of 10±1 mV for cells from ischemic cardiomyopathy and of 13±2 mV for cells from dilated cardiomyopathy. In voltage clamp mode the L-type Ca2+ current showed reversible decrease during stimulation at 1 and 2 Hz. Recovery from inactivation during a double pulse protocol was slow (75±3% at 500 ms, 89±3% at 1000 ms) and followed the decay of the [Ca2+]i transient. Conclusions: The negative force-frequency relation of the failing human heart is due to a decrease in Ca2+ release of the cardiac myocytes at frequencies ≥0.5 Hz, more pronounced in dilated than in ischemic cardiomyopathy. Inhibition of ICaL at higher frequencies, at least partially related to an increase in diastolic [Ca2+]i, will contribute to this negative staircase because of a decrease in the trigger for Ca2+ release, and of decreased loading of the SR.

KEYWORDS Heart failure; Humans; Single cells; Calcium; Calcium channel; Sarcoplasmic reticulum; Frequency potentiation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
In chronic heart failure the ability of the heart to maintain adequate output in the face of increased demand is reduced. While peripheral factors such as skeletal muscle dysfunction may contribute to the reduced exercise tolerance in patients with chronic heart failure [1], intrinsic contractile dysfunction of the cardiac muscle was demonstrated during studies of isolated muscle preparations. At low frequencies of stimulation, the peak developed tension was not decreased in ventricular muscle strips from failing human hearts [2, 3], but the rate of tension development and the rate of relaxation, were reported to be reduced [3], as well as the response to β-adrenergic stimulation [2]; cAMP content in failing hearts appeared to be reduced [4]. Also for single cardiac cells isolated from failing hearts, prolonged relaxation and reduced shortening amplitude at physiological stimulation rates, as well as a reduced response to β-adrenergic stimulation, were reported [5, 6]. Disturbances in Ca2+ homeostasis with slow diastolic Ca2+ removal and reduced Ca2+ responsiveness of the contractile elements were reported for multicellular preparations [3, 7]. In single cardiac cells isolated from failing hearts, it was found that the amplitude of the [Ca2+]i transient was reduced, most likely related to a decrease in Ca2+ release from the sarcoplasmic reticulum (SR) [8]. Decreased Ca2+ (re)uptake in the SR may be an important factor. Such a decreased SR Ca2+ uptake is supported by findings of a slower rate of decay of the [Ca2+]i transient [3, 8, 9], and of a reduced expression of the SR Ca2+ ATPase [10–13]; functional studies of Ca2+ uptake in SR membranes showed a reduced uptake, e.g. [13, 14], although some studies reported normal levels of Ca2+ ATPase activity [15]. In a recent study, inhibition of Ca2+ uptake in the SR reduced the difference between relaxation rates of myocytes from failing and non-failing hearts, consistent with the idea that in heart failure SR Ca2+ uptake is reduced [16]. Other factors may also contribute to a decrease in Ca2+ release from the SR, such as changes in the Ca2+ release channel, the ryanodine receptor, itself [17, 18]. Studies of the L-type Ca2+ channel did not reveal a decrease in current density [19, 20], although expression of the channel (dihydropyridine receptor) was reported to be reduced [10].

Human cardiac muscle from failing heart also shows a reversal of the contraction-frequency relation with a decrease in contractile performance at higher rates of stimulation, in contrast to normal cardiac muscle where contraction amplitude increases at higher frequencies [6, 21]. This reversal of the frequency-dependent behaviour has also been related to a reduction in the amplitude of the [Ca2+]i transient at higher frequencies of stimulation [22]. Reduced Ca2+ uptake by the SR in heart failure has been reported in association with the negative frequency relation [22], but the contribution of this and other cellular mechanisms have not yet been clarified. In the present study we have examined the frequency dependence of Ca2+ release from the SR in single cells isolated from human hearts with end-stage failure. We have investigated the role of membrane potential and of possible changes in the trigger for Ca2+ release which may contribute to the particular characteristics of the failing heart. Cells from hearts with either ischemic or dilated cardiomyopathy were studied and their properties compared.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Cell isolation from failing human heart
Single myocytes were isolated enzymatically from the hearts of patients with end-stage heart failure obtained at the time of transplantation. The procedure was approved by the Ethical Committee of the University Hospital and conform to the principles outlined in the Declaration of Helsinki. The patient characteristics are shown in Table 1. Five hearts were from patients with ischemic cardiomyopathy, six from patients with dilated cardiomyopathy. All patients were in severe symptomatic heart failure, with most patients in NYHA class IV at the time of admittance. In addition to measurement of the ejection fraction with isotope scintigraphy, decreased cardiac contractility was documented during echocardiography. All patients received standard therapy for chronic heart failure; 4 patients were on amiodarone as treatment for ventricular arrhythmias.


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  		Table 1 Patient data

 
The procedure for cell isolation was modified after Beuckelmann et al. [8]. The hearts were collected by us in the operating room at the time of surgery. After resection the heart was placed in ice-cold modified Tyrode's solution (in mM: NaCl 130, KCl 27, HEPES 6, MgSO4 1.2, KH2PO4 1.2, glucose 50; pH 7.20 with NaOH) and transported to the lab. A wedge of the left ventricular wall with its perfusing coronary artery was excised and the artery cannulated. If possible the left anterior descending coronary was used, otherwise the left circumflex or a posterior branch was used. The time between excision and cannulation was between 15 and 35 min, except for 3 hearts of which the aortic/pulmonary valve was prelevated (hearts kept on ice-cold cardioplegic solution) and for which time to cannulation was 40 to 85 min; this delay did not manifestly influence the success rate of the isolation procedure. After cannulation the tissue wedge was perfused (at 37°C) with a Ca2+ free Tyrode's solution (in mM: NaCl 130, KCl 5.4, HEPES 6, MgSO4 1.2, KH2PO4 1.2, glucose 20; pH 7.20 with NaOH) for 30' followed by enzyme perfusion for 40' (collagenase 1.4 mg/ml, Boehringer Mannheim, and protease type XIV 0.1 mg/ml, Sigma, in Ca2+ free solution). The enzyme solution was washed out with a low Ca2+ solution (Ca2+ free solution with 0.25 mM CaCl2 added) or with a modified Kraft–Brühe (KB) solution [23](in mM: KCl 30, KH2PO4 30, glutamic acid 50, taurine 20, MgSO4 3, EGTA 0.5, glucose 10; pH 7.30 with KOH). The tissue was then sectioned perpendicular to the wall in slices of 2–4 mm; of these slices the midmyocardial layers were dissected and minced in low Ca2+ or KB solution. The preparation was then filtered and resuspended in low Ca2+ or KB solution. After 20' for low Ca2+ solution, and after 2–3 h for KB solution, the solution was slowly replaced with a normal Tyrode's solution containing 1.8 mM CaCl2. Ca2+-tolerant cell yield was 5–15% for recovery in low Ca2+ solution, and 40–50% for recovery with KB solution. Cells were stored at room temperature and used within 18 h after isolation. When the 2 recovery solutions were used on cells isolated from the same hearts, we found that KB cells had similar action potential and current characteristics as low Ca2+ cells, but required prolonged stimulation before resuming contractile activity; [Ca2+]i transients slowly increased during this period, compatible with the hypothesis that these cells were Ca2+-depleted during storage in the KB solution. The majority of the results were obtained in cells recovered in low Ca2+ solution.

2.2 Voltage, current and [Ca2+]i recording
We used the whole-cell, ruptured patch clamp technique [24]. Membrane currents (voltage clamp mode) or membrane potential (current clamp mode) were recorded with an Axopatch 1D amplifier, filtered at 1 kHz and sampled and digitized at 4 kHz (Fastlb45, Indec Systems). [Ca2+]i was monitored with fluo-3 (60 µmol/l) or a combination of fluo-3 (30 µmol/l) and fura-red (70 µmol/l) [25–27]. The microscope was also equipped with a transmitted light source at 700 nm; a CCD camera made it possible to follow cell shortening visually on a TV monitor. With fluo-3 alone, fluorescence values were normalized for baseline fluorescence. For the combination of fluo-3 and fura-red we used calibration parameters which were obtained partly during in vitro calibration (β*Kd), partly during in vivo calibration (Rmax, Rmin) [25–27].

2.3 Solutions and experimental protocols
The pipette solution contained: (in mM) K-aspartate 120, KCl 20, K-HEPES 10, MgATP 5, MgCl2 0.5 (free [Mg2+] 0.8), NaCl 10, fluo-3 0.06 (or fluo-3 0.03 and fura-red 0.07); pH 7.20. The external solution contained: (in mM) NaCl 137, KCl 5.4, Na-HEPES 11.8, MgCl2 0.5, CaCl2 1.8, glucose 6; pH 7.35. With these solutions a junction potential of +10 mV occurred, which was not corrected for in the data presented, except for the values of resting membrane potential. All experiments were done at 36±1°C.

For 5 hearts a random sample of 20–30 cells from each heart, was measured with the aid of a calibrated eyepiece. The membrane potential, currents and [Ca2+]i transients were recorded in 2–5 cells from each heart.

Ca2+ release was studied during current clamp mode or voltage clamp mode. Action potentials were triggered by brief (3–6 ms) depolarizing current injections, applied at different frequencies. In voltage clamp mode holding potential was –70 mV; L-type Ca2+ current, ICaL, and Ca2+ release were recorded during 225 ms test depolarizations to +10 mV, preceded by a brief (75–150 ms) prepulse to –45 or –50 mV. The transient outward K+ current, Ito, was suppressed with 4-aminopyridine (2.5 mM) or in some cells in which this current was of low amplitude, by a prepulse to –40 mV. Different frequencies of stimulation were examined repeatedly and in random order.

To evaluate Ca2+ release, we measured [Ca2+]i at 20 ms after the depolarizing trigger pulse. This measurement will more closely reflect the flux of Ca2+ release than the peak [Ca2+]i value, since the flux is a function of the rate of rise of [Ca2+]i [28, 29]. Block by ryanodine (10 µmol/l) of the rapidly rising [Ca2+]i transients analyzed in the present study, confirmed that these transients indeed result from Ca2+ release from the SR.

For pooled data, means and standard errors are given. For statistical analysis we used an unpaired t-test for inter-group comparison, p<0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
3.1 Cell properties
Only Ca2+ tolerant cells with sharp edges and striations, and without cytoplasmic granulations were used. Cells were large with average cell length 191±5 µm and width of 39±2 µm (nhearts=5, ncells=100). The average membrane capacity was 196±14 pF (range 80–260 pF, n=31). All cells were well polarized with resting membrane potentials –78±1 mV (n=31) and contracted upon stimulation.

3.2 Frequency dependence of Ca2+ release
Fig. 1 shows an example of action potentials and [Ca2+]i transients recorded at 0.03, 0.25, 0.5, 1 and 2 Hz stimulation in a cell isolated from a patient with dilated cardiomyopathy. At all frequencies, the [Ca2+]i transients had a rapid upstroke, indicative of Ca2+ release from the SR. From 0.03 Hz to 0.5 Hz, the amplitude of the peak [Ca2+]i transient increased slightly; at 1 and 2 Hz, the peak value of [Ca2+]i increased further, but resting [Ca2+]i also increased. Therefore, if we measured the actual Ca2+ release, evaluated as the increase in [Ca2+]i at 20 ms after the depolarizing trigger, it decreased at 1 Hz and even more so at 2 Hz.


Figure 1
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  		Fig. 1 Action potentials and [Ca2+]i transients recorded at different frequencies of stimulation, after a steady state was reached. Cell isolated from a patient with dilated cardiomyopathy, capacity 260 pF.

 
The pooled results of a total of 31 cells are shown in Fig. 2. For the ischemic cardiomyopathy (nhearts=5, ncells=15), peak [Ca2+]i increased throughout the frequency range, although the increase was significant only for 2 Hz compared to 0.03 or 0.25 Hz. There was an increase in diastolic [Ca2+]i at 1 and 2 Hz (Fig. 2A, left). The actual release was maximal at either 0.5 or 1 Hz, and decreased at higher frequencies (Fig. 2 B, left). For the dilated cardiomyopathy (nhearts=6, ncells=16), peak [Ca2+]i was maximal at 0.25 Hz, and didn't increase with frequency; diastolic [Ca2+]i already increased slightly at 0.5 Hz and further increased at 1 and 2 Hz (Fig. 2A, right). The actual Ca2+ release was maximal at 0.25 Hz or 0.5 Hz, and markedly decreased at higher frequencies (Fig. 2B, right). A small number of cells was also tested at 3 Hz; at this frequency further decrease of Ca2+ release occurred.


Figure 2
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  		Fig. 2 Frequency dependence of [Ca2+]i transients. Pooled data of 15 cells from 5 hearts from patients with ischemic cardiomyopathy (CMP), and of 16 cells from 6 hearts from patients with dilated cardiomyopathy; data were obtained during steady state stimulation at different frequencies as illustrated in Fig. 1 with the lowest frequency at 0.03 Hz. A: Peak amplitude of the [Ca2+]i transient (solid triangle up), normalized (norm.) for the maximal value in each cell, and the change in resting resting [Ca2+]i (open triangle down) normalized for the resting value at 0.03 Hz. B: The actual Ca2+ release (solid circles), i.e. the increase in [Ca2+]i at 20 ms after depolarization, normalized for the maximal value for each cell.

 
In all cells [Ca2+]i transient had rapid upstroke with a time to peak of 37±2 ms at 0.5 Hz (n=31).

3.3 Action potential characteristics at different frequencies
In the example of Fig. 1 it can be seen that the action potential duration (APD) decreased with frequency. This was observed in all cells. Fig. 3A shows the pooled data for cells from ischemic cardiomyopathy, and for cells from dilated cardiomyopathy. In both groups APD decreased with frequency; there were no significant differences in APD between the groups. APD50 and the APD90 decreased to the same extent. Resting membrane potential did not change with frequency (not shown).


Figure 3
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  		Fig. 3 Action potential characteristics. Pooled data of 15 cells from 5 hearts from patients with ischemic cardiomyopathy, and of 16 cells from 6 hearts from patients with dilated cardiomyopathy. A: Action potential duration at different frequencies. The open symbols represent the action potential duration at 50% repolarization, APD50; solid symbols represent action potential duration at 90% repolarization, APD90. B: Changes of plateau potential with stimulation frequency. The potential was measured at 40 ms after the trigger pulse, or on the top of the dome for action potentials with pronounced spike-and-dome pattern. Individual data of cells are shown in dotted lines, mean values are shown in solid circles.

 
In addition to a decrease in duration, the membrane potential at the plateau decreased with frequency (see Fig. 1), although not to the same extent in all cells. Plateau potential was measured at 40 ms after the trigger pulse, or, in the case of cells with a pronounced spike and dome pattern of the action potential, on the top of the dome. The individual and pooled data are shown in the Fig. 3B. In cells from ischemic cardiomyopathy the plateau voltage dropped by 10±1 mV between 0.03 to 2 Hz, versus by 13±2 mV in cells from dilated cardiomyopathy (p=NS). Cells with a drop of more than 15 mV were more frequently found in the dilated hearts (7/16 cells, versus 3/15 cells for ischemic hearts). These cells had the most pronounced decrease in Ca2+ release with increased frequency of stimulation.

Both the decrease in action potential duration as well as the drop in the plateau voltage will promote loss of Ca2+ from the cell through the Na/Ca exchanger and contribute to a decrease in Ca2+ load of the SR. The loss of plateau voltage could possibly be explained by a loss of ICaL and we therefore further examined Ca2+ release and ICaL in voltage clamp mode.

3.4 Frequency-dependent behaviour of ICaL
We first examined whether stimulation with a square pulse of fixed duration and amplitude would alter the frequency dependence of Ca2+ release. An example is shown in Fig. 4. Panel A shows action potentials and [Ca2+]i transients at the indicated frequencies and illustrates the maximal release at 1 Hz with decrease at 2 Hz, together with an increase in diastolic [Ca2+]i and a drop in action potential duration and plateau voltage. In the lower panels, the same cell was stimulated in voltage clamp mode with square pulses from –70 to +20 mV of 225 ms duration. The decrease of Ca2+ release at 2 Hz is slightly less, but is not reversed.


Figure 4
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  		Fig. 4 Comparison of Ca2+ release during action potentials to Ca2+ release during square voltage clamp pulses of fixed duration. A: Ca2+ release during action potentials at the indicated frequencies; fluorescence signals denoting the [Ca2+]i transients are normalized to the baseline fluorescence before the start of stimulation, and are expressed in arbitrary units, a.u. B: Ca2+ release in the same cell during 225 ms square pulses from –70 to +20 mV at the indicated frequencies. Cell isolated from a patient with ischemic cardiomyopathy, capacity 240 pF.

 
During this kind of protocol the behaviour of the Ca2+ current can not be studied because of the presence of INa and of the transient outward current. We therefore next examined the frequency behaviour of ICaL and Ca2+ release during steps preceded by a conditioning pulse to –40 mV to inactivate INa and the transient outward current (for some experiments 2.5 mM of 4-aminopyridine was also added); the test potential was +10 or +20 mV. These steps were repeated at different frequencies, and in between the cell was rested for 45 s at the holding voltage of –70 mV. Fig. 5, A and B, illustrates the results of such a protocol for a cell from a patient with ischemic cardiomyopathy and one from a patient with dilated cardiomyopathy, and shows the amplitudes of ICaL and the [Ca2+]i transients at the indicated frequencies. There is a pronounced decrease in the amplitude of ICaL at 2 Hz; at the same time there is an increase in diastolic [Ca2+]i. The inhibition of ICaL was directly related to the frequency of stimulation, being largest at 2 Hz. This frequency-dependent inhibition was always reversible; different frequencies were tested randomly and repeatedly, yielding similar results. Because we are recording in K+-containing solutions we have to exclude that the decrease in peak inward current could be due to a frequency-dependent increase in a ultra-rapidly activating outward current, IKur [30]. However, such current would be blocked by 4-aminopyridine, and in addition, after block of ICaL with nifedipine or nisoldipine, high-frequency stimulation did not reveal a frequency-dependent increase in outward current (n=2), making it unlikely that IKur is involved. The frequency-dependent decrease of ICaL was examined in a total of 15 cells. Fig. 5C shows the decrease in ICaL during stimulation at 1 Hz, expressed as the amplitude of ICaL at 1 Hz relative to the amplitude of ICaL at 0.25 Hz. The data are plotted as a function of the decrease in plateau potential at 1 Hz frequency compared to 0.25 Hz (solid symbols are cells from dilated cardiomyopathy, open symbols from ischemic cardiomyopathy). These data illustrate two important points. First, there appears to be a direct relation between the decrease in action potential plateau and the decrease in ICaL; linear regression analysis of the pooled data yields a r=0.9 with P<0.001. Second, the decrease in ICaL with frequency varies from mild to severe between cells, with the most pronounced inhibition observed in cells from dilated cardiomyopathy. In none of the cells studied did stimulation with fixed pulse duration reverse the negative frequency behaviour.


Figure 5
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  		Fig. 5 Frequency-dependent inhibition of ICaL. A: Cell from a patient with ischemic CMP. The cell was stimulated with 225 ms square pulses from –70 mV to +10 mV, preceded by a 75 ms pulse to –40 mV to inactivate INa; 2.5 mM 4 aminopyridine was present to block the transient outward current. The [Ca2+]i transients represent the steady state at the indicated frequency. For 2 Hz, the current record at the onset of the stimulation is shown in dashed lines; for 0.5 Hz, the current at onset and steady state superimpose. B: Cell from a patient with dilated CMP. The cell was stimulated with square pulses from –70 mV to +20 mV, preceded by a 75 ms pulse to –40 mV to inactivate INa. The [Ca2+]i transients were obtained at steady state at the indicated frequency; the dashed line is the current at the onset of stimulation; for 0.5 Hz, the change is small. C: Relation between the decrease in action potential plateau, measured as the change in plateau potential from 0.25 Hz to 1 Hz, and the frequency–dependent inhibition of ICaL, measured as the amplitude of ICaL at 1 Hz expressed as a percentage of the amplitude of ICaL at 0.25 Hz. Solid symbols are data obtained in cells from dilated CMP (4 hearts), open symbols are cells from ischemic hearts (3 hearts).

 
One possible reason for the decrease of peak ICaL with frequency is slow recovery from inactivation. We examined this in 6 cells; a representative example is shown in Fig. 6, obtained from the same cell as Fig. 5B. Recovery of ICaL was studied in a 2-step protocol with increasing duration between the reference and test pulse. Fig. 6 shows that recovery of ICaL paralleled the decay of the [Ca2+]i transient. The time for full recovery of ICaL exceeded 1 s, and coincided with full recovery of Ca2+ release. In a total of 6 cells, recovery of ICaL at 500 ms was 75±3%, at 1000 ms 89±3%.


Figure 6
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  		Fig. 6 Time course of recovery of ICaL. The holding voltage was –70 mV; after a 150 ms prepulse to –45 mV ICaL was recorded during a 300 ms step to +10 mV; the membrane is repolarized to –70 mV and the recovery of ICaL is examined during test steps to +10 mV (preceded by a 150 ms prepulse to –40 mV) after increasing time intervals. The capacity currents on repolarization have been erased for clarity. Cell from a patient with dilated cardiomyopathy, same cell as Fig. 5B.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
4.1 Ca2+ release decreases at frequencies above 0.5 Hz
The existence of a negative force-frequency relation in failing human cardiac muscle was demonstrated first at 30°C [31]. The relevance of this finding was confirmed in experiments at more physiological temperatures and frequencies by Mulieri et al. [21]. In a more recent study in multicellular preparations force measurements were complemented with measurements of [Ca2+]i transients at physiological temperature [22]. While in multicellular preparations, the contribution of changes in metabolic supply or of non-myocyte components, can never be completely excluded, studies on single cells are free of these possibly confounding variables. Measurements of shortening in intact single cells had also shown a decrease in contraction at higher frequencies of stimulation [6, 32].

Our data confirm the hypothesis that the decrease in force of contraction at frequencies above 0.5 to 1 Hz is due to a disturbance in [Ca2+]i regulation [22, 33, 34], and we show that this is an intrinsic property of the myocytes of failing hearts. Our data indicate that the decrease in the amplitude of the [Ca2+]i transient is due to a decrease in Ca2+ release from the SR. Although myocytes from both ischemic and dilated cardiomyopathy showed the decreased Ca2+ release at frequencies ≥1 Hz, the negative slope was more pronounced for the dilated cardiomyopathy with an optimal Ca2+ release at lower frequencies of 0.25 or 0.5 Hz. These results are in agreement with the findings of Pieske et al. in dilated cardiomyopathy [22]. However, in our study we noted a significant increase in diastolic [Ca2+]i at higher frequencies of stimulation not described before [22]. This can be ascribed to the different properties of our fluorescent [Ca2+]i indicator, which is more sensitive to low [Ca2+]i than aequorin. Such an increase in diastolic [Ca2+]i can contribute to the diastolic dysfunction often observed in heart failure, and may also be critically involved in the regulation of Ca2+ release at these higher frequencies (see below).

4.2 Mechanisms of decrease of Ca2+ release with frequency
In our study of single myocytes we have been able to examine some possible underlying mechanisms of the negative force-frequency relation, which can not be studied in multicellular preparations. During simultaneous recording of action potentials and [Ca2+]i transients, we found a pronounced decrease in action potential duration, with a loss of plateau potential at higher frequencies of stimulation. Such a decrease in action potential duration was also described in multicellular preparations [22]. The values we find for APD90 are longer than reported for multicellular preparations [22, 35, 36], as can be expected for whole-cell recording versus the microelectrode technique. In single cells, only Beuckelmann and coworkers have studied action potentials [8, 37]. Our values are less than this group first reported in myocytes without [Ca2+]i buffering [8]; a lower internal Cl- concentration in our study could explain some of these differences. The decrease in action potential duration and in plateau voltage at higher frequencies of stimulation may contribute to, but apparently is not the (only) cause of, the decrease in Ca2+ release at higher frequences, since a negative frequency behaviour was also observed during stimulation with square pulses of fixed duration and amplitude.

The observed changes in action potential plateau voltage appear to be related to changes in ICaL with increased frequency. In all cells studied we found a decrease in amplitude of ICaL at higher rates of stimulation. This has not been described before, but most of the previous studies of ICaL in human ventricular myocytes have been performed at low frequencies of stimulation, and have not examined dynamic behaviour of ICaL [8, 19, 20, 38–40]. In addition, [Ca2+]i was usually buffered (except for [8, 39, 38]). One study of frequency-dependent changes of ICaL in human myocytes, reported a frequency-dependent upregulation of ICaL in atrial myocytes of non-failing hearts, but absence of such an effect in left ventricular myocytes from failing hearts [41]. Although a frequency-dependent decrease was not observed in this study, the data reported in [41]may actually reflect similar properties of failing myocytes as observed in our study, but less pronounced due to [Ca2+]i buffering, and other differences in experimental conditions (room temperature, Na-free solutions).

Slow recovery of inactivation may account for the loss of ICaL at higher frequencies of stimulation. Even a modest degree of incomplete recovery at 1000 ms will during repeated stimulation at 1 Hz, and certainly at 2 Hz, accumulate and lead to significant depression of peak ICaL. Recovery from inactivation has been described only in the study by Benitah et al. [40]on myocytes from hypertrophied hearts in which a component of very slow recovery was also found. Inactivation of the cardiac L-type Ca2+ channel is due to both voltage- and Ca2+-dependent mechanisms [42]. At this time we can not distinguish whether the slow recovery is from voltage-dependent inactivation or related to Ca2+-dependent inactivation. Presence of the latter was demonstrated for Ca2+ entry through the Ca2+ channel in human myocytes [19]. An important component of Ca2+-dependent inactivation may be related to the increase in diastolic [Ca2+]i at higher frequencies of stimulation; the parallel time course of recovery of [Ca2+]i and decay of [Ca2+]i may be related to such a process. However, the decrease in ICaL was not always proportionate to rise in diastolic [Ca2+]i. One explanation is that local [Ca2+]i is higher and induces more inactivation, as we have previously observed in guinea pig ventricular myocytes [43]. Another possibility is that a voltage-dependent mechanism also contributes. The experiments by Benitah et al. [40]were performed with [Ca2+]i buffering and therefore seem to point towards a voltage-dependent mechanism.

The decrease of ICaL with frequency will be an important factor in the negative force-frequency behaviour as it will decrease the trigger for Ca2+ release [8]as well as the net Ca2+ influx into the cell, directly and through the effect on action potential duration and plateau levels. Negative force-frequency behaviour has previously been associated with the decreased Ca2+ uptake into the SR [10, 12–14, 44]. Our finding of a rise in diastolic [Ca2+]i at higher frequencies is additional evidence for a decrease in Ca2+ removal from the cytoplasm, very likely by the decrease in SR Ca2+ uptake. However, while decreased SR uptake explains the lack of an increase in force with frequency, it does not explain the actual loss of force, and as we have shown, of Ca2+ release at higher frequencies. Our finding of a decrease in ICaL is the first such rate-dependent mechanism which can explain the actual decrease in Ca2+ release at higher frequencies. The present findings obviously do not exclude that yet other mechanisms may contribute to the disturbed [Ca2+]i homeostasis, as e.g. changes in the Ca2+ release channel [17, 18]and in Na/Ca exchange [12, 44, 45]. In rat ventricular muscle studied in the lower frequency range up to 2 Hz, a negative force-frequency relation has been ascribed to high [Na+]i in combination with short action potentials [46, 47]. We also recently reported that high [Na+]i could reverse force-frequency relations [27]. At present no data on [Na+]i in failing human cardiac muscle are available, but contractions and [Ca2+]i transients at very low frequencies are not potentiated, as would be expected for high [Na+]i.

4.3 Limitations of the study
Our data were not compared to similar experiments performed on normal human cardiac cells. While such comparison would be very valuable, true normal human cardiac muscle is difficult to obtain. Biopsies obtained during cardiac surgery are almost always from diseased muscle, while donor hearts which are not used for transplantation, are rare. Our approach has therefore rather been to investigate the mechanisms proper to the myocytes isolated from (proven) failing hearts. Such results are directly relevant to the function of these hearts.

Patients with chronic heart failure are on multiple-drug therapy. The present data do not allow the study of possible drug-related effects, because most of the patients have similar therapy, and the numbers therefore are too small. While most of the drugs should be cleared from the tissues during the isolation procedure, long-term drug effects can not be excluded. Especially for amiodarone, we checked whether the changes in ICaL with frequency could be correlated with amiodarone treatment. This was not the case; cells with the most pronounced frequency-dependent inhibition came from patients 3 and 5, who were not on amiodarone treatment.

Intact single cells have retained the properties of the intact muscle, but are a small sample of the heart. Variability between cells exists, and probably represents true variability in vivo. Especially in the ischemic cardiomyopathy, not all cells have been subjected to the same stimulus for remodeling, such as ischemia or increased wall stress. This can be considered a limiting factor, but need not be if appropriate samples are studied. The major contribution of studies on single cells comes from the possibility to study subcellular mechanism which can not be evaluated in the multicellular preparation as was demonstrated in the present study. These results can then be integrated with the data obtained by other methods.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
We have shown that in myocytes from failing hearts Ca2+ release from the SR decreases with stimulation frequencies above 0.5 Hz. Cells from dilated cardiomyopathy appear to have a more pronounced negative [Ca2+]i-frequency relation than cells from ischemic cardiomyopathy. Diastolic [Ca2+]i increases at 1 and 2 Hz probably reflecting decreased Ca2+ uptake in the SR. The decrease in Ca2+ release is at least in part due to a rate-dependent decrease in ICaL, which shows slow recovery from inactivation. Our findings of a decrease of Ca2+ release with frequency and the link to a decrease in ICaL may help to explain the beneficial effect of rate-lowering drugs such as β blockers, and digitalis, in chronic heart failure. Lower heart rates will not only improve diastolic function but also systolic function.

Time for primary review 40 days.


   Acknowledgements
 
The study was supported by a grant from the Bekales Foundation (K.R.S.). K.R.S. is a postdoctoral researcher at the FWO, the Belgian Fund for Scientific Research. The authors acknowledge the major contribution of Prof. K. Mubagwa as the instigator and coordinator of the human heart studies. We thank Dr. M. Stengl for his help in cell isolation, and all the members of the Divisions of Cardiology and Cardiac Surgery involved in the transplantation program for their invaluable collaboration.


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

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