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Cardiovascular Research 1998 37(2):445-455; doi:10.1016/S0008-6363(97)00257-5
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Single-channel properties of L-type calcium channels from failing human ventricle

Renate Handrocka, Frank Schrödera, Stephan Hirtb, Axel Haverichc, Clemens Mittmannd and Stefan Herziga,*

aDepartment of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Cologne, Germany
bDepartment of Cardiothoracic Surgery, University of Kiel, Kiel, Germany
cDepartment of Cardiothoracic Surgery, Medical School of Hannover, Hannover, Germany
dDepartment of Pharmacology, University of Hamburg, Hamburg, Germany

* Corresponding author. Tel. (+49-221) 478-6064; Fax (+49-221) 478-5022.

Received 13 June 1997; accepted 9 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of our study was to analyse the single-channel properties of L-type calcium channels from failing human heart and to compare them to the respective animal data. Furthermore, we intended to evaluate the feasibility of future single-channel studies on the role of calcium channels in the pathophysiology of heart failure. Methods: Single L-type calcium channels were recorded in ventricular myocytes from explanted failing human heart, using the cell-attached configuration of the patch-clamp technique. Results: One or more successful registrations of calcium channels could be obtained in 11 of 19 cell isolations. Determination of single-channel conductance yielded a mean value of 16.6±1.2 pS (70 mM Ba2+ as the charge carrier) under control conditions and 23.7±2.8 pS in presence of the calcium-channel agonist FPL 64176. The rapid gating process could be described by a C{leftrightarrow}C{leftrightarrow}O gating scheme. Slow gating analysis revealed a highly significant clustering of active and non-active sweeps. Conclusion: Single-channel measurements of L-type calcium channels in human failing ventricle are feasible and reproducible despite the varying patient characteristics. Their channel properties are qualitatively comparable to those found in other mammals. Whether there are quantitative differences due to the underlying heart failure can be elucidated in further studies.

KEYWORDS Calcium channel, L type; Single-channel recording; Heart failure; Human ventricular myocyte


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
L-type calcium channels are important for cardiac excitation–contraction coupling and the question arises whether they are affected in heart failure. A number of groups have addressed this issue, using the whole-cell configuration of the patch-clamp technique for measurement of the standard properties of L-type channels in human ventricular myocytes from terminally failing hearts [2, 3, 25]. In summary, all these properties of human channels are very reminiscent of those found in animal experiments, and a comparison between cells from failing ventricles and from non-failing donor ventricles has not revealed any major differences regarding current density or biophysics of the channels [25]. These findings, together with recent evidence that the prominent changes in calcium metabolism of failing human cardiomyocytes [4], may be caused by alterations of SR proteins ([13], but see also [22, 41]) or Na–Ca exchanger [10, 36, 43], suggest that the L-type channel is only of minor — if of any — importance for the cellular pathophysiology of heart failure.

However, one has to consider that a whole-cell current I is a function of several variables, being described at any time point as


Formula

where N is the number of functional channels in the cell, i is the single-channel current amplitude, factive is the fraction of available channels, i.e. those which actually participate in channel activity during a particular time period (e.g. a voltage step), and popen is the probability of the available channels to be in the open state. The number of L-type channels in heart failure (N) has been studied by non-electrophysiological techniques (dihydropyridine binding studies and quantification of messenger RNA). Apparently depending on the methods used, choice of species and form of failure, increases [47], decreases [44], or no effect on expression [40]were found. Thus, no firm conclusion regarding N can be drawn at the present moment. It is known from single-channel studies that both factive and popen are far below 100% even under conditions which stimulate channel activity to its maximum [7, 14, 31, 50], such that — even if N and i were known exactly — a given value for I could reflect vastly different channel properties regarding factive and popen.

Therefore, it is premature to conclude just from whole-cell current recordings that cardiac L-type channels are unaltered in congestive heart failure. Single-channel studies are required to test whether L-type channels play a role in the pathophysiology of failing human myocardium. However, there has not been any report of single-channel studies from human ventricle, despite the fact that single L-type channels can be recorded from ventricles of guinea-pig, rat, rabbit, chick, etc. [7, 32, 39, 45, 49], and from human atrial myocytes [20]. Furthermore, no single L-type calcium channels have been measured in any — either animal or human — model of cardiac pathology so far. Thus, before a detailed comparison of calcium-channel properties from healthy and failing human hearts is started, it is reasonable and necessary to test the feasibility of such a study. Therefore, we thoroughly investigated single human L-type channels from failing hearts. We tested the success rate for getting a consistent amount of viable single cells tolerant to a high Ba2+ concentration in the pipette, sufficiently long recording time, and reproducible data despite the varying patient characteristics. We focused our analysis on the characterization of the standard properties of single L-type calcium channels like conductance, voltage-dependence, fast and slow gating. We compared our data to those from animal ventricular myocytes and human atrial myocytes in order to check on qualitative agreement or gross abnormalities in single-channel behaviour.

This first single-channel study on human ventricular L-type channels highlights that an analysis of single-channel properties in this tissue is feasible and in qualitative agreement with animal data. On the basis of these results, a systematic comparison of failing and non-failing human myocytes in the future will be possible.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of cardiomyocytes
Ventricular myocytes were prepared from hearts of patients with end-stage heart failure caused by ischemic or dilated cardiomyopathy who were undergoing transplantation. Immediately after excision of tissue from the left ventricular wall, the samples were placed in a preoxygenated solution of 4°C (solution A) composed of (mM) NaCl 100, KCl 10, MgSO4 5, dextrose 20, taurine 50, MOPS 5 (pH 7.4) and taken to the laboratory within 15 min. After removal of fat and connective tissue, the ventricular tissue was dissected into small slices of about 2x2x0.5 mm in oxygenated solution A at room temperature. The pieces were enzymatically digested in 10 ml of solution A in the presence of collagenase (1.5 mg/ml, type CLS 1, Worthington Biochemical Cooperation, NJ), trypsin (1 mg/ml, type III, Sigma, Germany) and bovine serum albumine (BSA, 10 mg/ml, Sigma, Germany) at 37°C for 40 min under continuous stirring and shaking. After decanting the supernatant and washing the tissue with solution A once, a second incubation of variable duration (60.9±5.1 min, ranging from 43 to 83 min) in the presence of collagenase (0.5 mg/ml) and BSA (1 mg/ml) followed. Samples (40 µl) of cell suspension were taken every 10 min for morphological control at a 100x and 400x magnification. When the density of elongated and cross-striated cells remained stable for two or three consecutive samples, the process was stopped by diluting the cell suspension with solution A (1:5, 37°C). After 15 min of sedimentation, the supernatant was replaced by a solution (solution B) containing (mM) K-glutamate 50, KCl 40, KH2PO4 20, taurine 20, KOH 20, MgCl2 3, HEPES 10, EGTA 5, dextrose 10 (pH 7.4). Ventricular cells were disaggregated by gentle mechanical agitation and then stored at 4°C for 1 h.

The investigation conforms with the principles outlined in the Declaration of Helsinki and was approved by a local ethics committee.

2.2 Measurement of calcium channels
For measurement of L-type calcium-channel currents, cells were placed in disposable 2-ml perfusion chambers containing (mM) K-glutamate 120, KCl 25, MgCl2 2, HEPES 10, EGTA 2, CaCl2 1, Na-ATP 1, dextrose 10 (pH 7.4 with NaOH, 21–23°C). Pipettes (borosilicate glass, 7–10 M{Omega}) were filled with (mM) BaCl2 70, sucrose 110, HEPES 10 (pH 7.4 with TEA-OH). Single calcium channels were recorded in the cell-attached configuration of the patch-clamp technique [11]. Barium currents were elicited by depolarising test pulses of 150 ms at 1.66 Hz, recorded at 10 kHz and filtered at 2 kHz (–3 dB, 4-pole Bessel) using an Axopatch 200A (Axon Instruments, Foster City, CA). The pClamp software (version 6.0, Axon Instruments) was used for data acquisition and analysis.

FPL 64176 (methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate, RBI, Natick, USA) was added in a 20-µl volume of a 0.1 or 1 mM solution (prepared from a 10 mM stock solution in dimethylsulfoxide (DMSO)) to the dish, yielding final drug concentrations between 0.8 and 8 µM and <1% DMSO.

2.3 Data analysis
Linear leak and capacity currents were digitally subtracted using the average currents of non-active sweeps. Openings and closures were identified by the half-height criterion using pClamp software. Activation and inactivation data were fitted to a Boltzmann function according to the equation


Formula

where V is the membrane potential, V0.5 the potential at which activation or inactivation is half-maximal, and k the slope factor describing the steepness of the curve.

All averaged values are given as means±SEM.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Isolated ventricular myocytes
A summary of the patient characteristics of those hearts where calcium-channel activity could be measured is shown in Table 1. The age of the patients (9 men, 2 women) was 49.9±2.9 years. End-stage heart failure was caused by ischemic (n=7), dilated (n=3) cardiomyopathy or vasculopathy (n=1). The latter was a cardiac transplant atherosclerosis due to the first transplantation in 1988. Cardiac index where available ranged from 1.4 to 3.2 l/min/m2 and ejection fraction from 25 to 52%, but the values were determined some months before heart transplantation in most cases. The actual medication at the time of transplantation related to heart failure is listed in Table 1; the medication, which is subsumed under ‘other’ drugs, refers to additional non-cardiovascular diseases.


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  		Table 1 Patient characteristics of those hearts which gave successful calcium-channel recordings

 
The living cell yield was about 5–10% but could be much higher or lower in single cases. Only cells with clear cross-striations and without spontaneous contractions were selected for experiments.

All together, 19 cell isolations were done for this study, and in 11 cases we have been successful in measuring calcium-channel activity. In these experiments, 25–50 G{Omega} seals per cell isolation were obtained during the 5–8-h recording period. In the case of the other eight hearts, no currents could be recorded. This was predominantly due to the inability of getting stable seals or seals at all, to bad cell quality or a small cell yield. Only in a few cases were there no channels in the patches in spite of getting numerous stable seals. The time passing between explantation and receipt of the tissue was less than 30 min in the majority of cases. Otherwise, tissue was kept in ice-cold cardioplegic solution for as long as 12–17 h. We could not find any obvious correlation between the length of this period and the cell yield, cell quality or calcium-channel activity. These parameters also appeared not to depend on the type of cardiomyopathy.

3.2 Single-channel conductance, voltage-dependence of activation and inactivation
Single L-type calcium channels from failing human heart were characterized by their conductance and voltage dependence of activation and inactivation. Fig. 1A shows original traces with single-channel activity, elicited by 150 ms depolarising pulses from a holding potential of –100 mV to test potentials between –20 and +30 mV. Channel availability and open probability increased with voltage. We determined the conductance by plotting the amplitude of apparently fully resolved openings against the test potential for every single experiment, as it is shown in Fig. 1B. The mean of the slopes of the linear relationships is 16.6±1.2 pS. This low value may be short of the true conductance properties due to an underestimation of amplitude at negative test potentials. In order to facilitate the occurrence of fully resolved openings at all investigated test potentials, we induced long openings by applying the calcium-channel agonist FPL64176 (0.8–8 µM) to the bath. In 4 of 5 experiments, we were successful in stimulating the calcium-channel activity, as it is exemplified by the original current traces at different test potentials in Fig. 1C. In three of these experiments, long openings could be analysed at different test potentials. The slopes of the linear i/V relationships were 29.3 (data points of this experiment included in Fig. 1B), 20.5 and 21.3 pS. The mean conductance was 23.7±2.8 pS.


Figure 1
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  		Fig. 1 Ba2+ currents through a single cardiac L-type calcium channel at different test potentials and the deduced i–V relationship. A: Ca2+ channel activity elicited by 150 ms test pulses from –100 mV to the indicated test potentials. Scale bars: 20 ms and 2 pA. B: The single-channel amplitude of six recordings under control conditions and one experiment in presence of FPL64176 ({blacksquare}) is plotted as a function of the test potential. The protocol depicted in (A) was used. Each symbol represents one experiment. A mean single-channel conductance of 16.6 pS was determined by linear regression for the control data. The linear regression of the values in presence of FPL64176 yielded a conductance of 29.3 pS. For the original traces of this experiment see (C). C: Ca2+ channel activity induced by depolarisations from –100 mV to the indicated test potentials in the presence of 8 µM FPL64176. Scale bars: 20 ms and 1.5 pA.

 
Fig. 2 shows the voltage dependence of activation and inactivation. Depolarising pulses of 150 ms duration were elicited either from a given holding potential of –100 mV to the indicated test potentials (activation) or from decreasing holding potentials to a constant test potential of +20 mV (inactivation). Series of at least 60 pulses were used for each voltage. The fraction of active sweeps was checked as a function of the test and holding potentials in five patches each. Activation and inactivation curves could be described by a Boltzmann function with a half-maximal activation at –3.5 mV (slope 4.9 mV) and a half-maximal inactivation at –60 mV (slope –7.5 mV). Maximum availability was about 50% in both cases and could be achieved by test pulses from –100 to +20 mV. Therefore, we continued our study by using this protocol.


Figure 2
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  		Fig. 2 Voltage-dependence of activation and inactivation. Activation curve: channel availability was determined from five experiments recorded as in Fig. 1A shown and plotted against the test potential. The data were fitted by a Boltzmann distribution (see Section 2), yielding the following values: V0.5=–3.5 mV, k=4.9 mV. Inactivation curve: channel availability was calculated from five experiments, recorded by 150 ms depolarisations from the indicated holding potentials to a test potential of +20 mV. Again, a Boltzmann function was fitted to the data, resulting in the following values: V0.5=–60 mV, k=–7.5 mV.

 
3.3 Rapid gating analysis
Long series of depolarisations (465±58 sweeps) from –100 to +20 mV were obtained in five single- and three double-channel patches. In Fig. 3, the gating of a human L-type calcium channel is exemplified together with the ensemble average current of 600 consecutive depolarisations. We determined the average values for the standard gating parameters for all of these experiments, as indicated in Table 2. Values from double-channel patches were corrected for the number of detected channels where applicable. The availability, calculated as the fraction of active sweeps, was 39.5±5.5% and the open probability of the active recordings was 10.5±3.6%. The mean life time of the open state was 0.55±0.06 ms. As the mean closed time cannot be corrected for the number of recorded channels, only single-channel patches were included in the average value of 5.53±1.14 ms. The same holds true for the mean first latency (23.3±2.4 ms) which reflects the period after depolarisation which passes until the first opening occurs. The ensemble average current reached its maximum at 37.9±13.4 fA. Inactivation of the ensemble current (29.1±9.5%) was quantified after 150 ms of depolarisation.


Figure 3
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  		Fig. 3 Ba2+ currents through a single cardiac L-type channel. Ba2+ currents were stimulated by 150 ms step depolarisations from –100 to +20 mV. At the bottom, the ensemble average current, determined from 600 consecutive sweeps, is depicted (patient A.C.). Scale bars denote 20 ms and 2 pA (single traces) or 20 fA (ensemble average).

 

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  		Table 2 Summary of the fast gating parameters from five or eight experiments of this study with long-lasting recordings at a test potential of +20 mV (second column)

 
Rapid gating was further characterized by histogram analysis. Therefore, the number of openings or closures during an experiment was plotted against the open or closed time, respectively. Fig. 4 illustrates such histograms of one experiment. Results obtained for individual channels were similar to those obtained by pooling all data. Open time histograms were analysed by fitting a monoexponential function to the data of each experiment and by averaging the time constants, yielding a mean open time constant {tau}open of 0.43±0.04 ms. Closed time distribution was described by a biexponential process, giving a mean fast time constant {tau}f, closed of 0.31±0.09 ms and a mean slow time constant {tau}s, closed of 8.71±1.45 ms. The proportion of fast and slow closed time constants to the total histogram was nearly equal. At the bottom of Fig. 4, the number of first openings is cumulatively plotted as a function of the latency. The function saturates over a period of 150 ms.


Figure 4
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  		Fig. 4 Rapid gating analysis. Open and closed time histogram of a single experiment (patient E.H.). The open time histogram follows a monoexponential distribution, and the closed time histogramm a biexponential one. The respective histograms of the pooled data gave a similar picture (not shown). The graph at the bottom shows the cumulative first latency distribution of the same experiment.

 
3.4 Slow gating analysis
Determination of the fast gating behaviour is an essential part for characterization of the electrophysiological properties of single-channel currents. Furthermore, it is also important to analyse the slow gating, i.e. the transition between available (active sweeps) and unavailable (blank sweep) channel states. Having a closer look at the original current traces in Fig. 3, it becomes obvious that there was a tendency for active and blank sweeps to appear in clusters. In order to get more insight in the mechanism underlying this transition, slow gating was analysed kinetically by the use of sweep histogram analysis (e.g. [14, 31]). Hereby, the number of consecutively active or blank sweeps is counted as an active or blank run and plotted as a function of the run length (given as ≥sweeps), yielding histograms of run duration as they are shown in Fig. 5. The active run histogram always followed a monoexponential distribution. By means of this exponential fit, time constants from five single-channel patches were calculated. The dimension of the time constants (no. of sweeps) was then converted into seconds by multiplying the number of sweeps in a run with 0.6 s. The average of these five time constants resulted in a mean value of 1.58±0.26 s (Table 3). This value clearly exceeds the test pulse interval of 0.6 s, suggesting that the availability is controlled by a long-lasting process. Blank run histograms could be fitted by a monoexponential (n=2) or biexponential (n=3) function, indicating in the latter case the presence of at least two unavailable states. Therefore, the mean blank duration was calculated from five experiments simply by multiplying the average number of adjacent blank sweeps with 0.6 s, yielding a value of 3.63±0.76 s.


Figure 5
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  		Fig. 5 Slow gating analysis. The duration of continuously active or non-active sweeps, an active or blank run, was determined by the use of sweep histogram analysis. The number of active or blank runs is plotted as a function of the run length from a single experiment (patient A.C.). The active run histogram was monoexponential, yielding a {tau}=2.53 s. The blank run data were fitted by a biexponential. The mean duration of the blank runs amounted to 2.17 s.

 

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  		Table 3 Summary of the slow gating parameters of five single channel patches

 
In order to investigate whether such runs of consecutively active or blank sweeps occur non-randomly, we used a runs analysis [18]. Hereby, the randomness is described by a standardized random variable Z which can be computed considering the number of runs, the total number of sweeps and the overall availability factive. A Z-value ≥2 indicates a significant degree of non-randomness. In each of our five experiments the Z-values were markedly above 2 (see Table 3), indicating that runs of consecutive active or blank sweeps occurred significantly clustered.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
This study shows for the first time that single-channel recordings allow a systematic characterization of the biophysical properties of L-type calcium channels in ventricular myocytes from failing human heart. Although the cell yield per cell isolation is markedly lower than what we experienced with guinea-pig myocytes — possibly due to the cardiopathy — the rate in successful channel recordings is comparable between guinea-pig and human failing ventricular myocytes. In contrast to our own preliminary experience with human atrial myocytes, where under similar conditions channels are regularly present in every third or fourth patch (unpublished data), it may last a whole experimental day to find a calcium channel in a particular preparation from failing heart.

The recordings were made under conditions specifically designed for calcium channels [50]. Indeed, we never found channels which did not conform to the criteria defined below for L-type calcium channels. In particular, T-type channels were not detected, despite the fact that we occasionally see T-type channel activity under the same recording conditions in guinea-pig and rat ventricular myocytes (unpublished data). This finding may be important with respect to recent data suggesting a role of the T-type calcium current in cardiomyopathic hamster heart [42]. However, the recovery of T-type channels may be a problem of the cell isolation procedure (for discussion, see [34]), and the fact that T-type channels were not observed is by no means proof of their absence in the native tissue.

The following criteria were applied and fulfilled in this study to confirm that the channel activity recorded (with 70 mM Ba2+ as charge carrier in the pipette) was indeed L-type [29].

First, the channel activity was long-lasting and persisted — almost stable — throughout the 150 ms voltage step (see Figs. 1 and 3Go).

Second, the voltage dependence of activation and inactivation (Fig. 2) is comparable to previous work with single L-type channels [23, 24]. The maximum availability in both the activation and inactivation curve is below unity. Concerning the inactivation, this is in agreement with previous results [7, 21, 31]. Although these show a rather broad variability in the maximum availability with values between 40% and 80%, unity is consistently never reached. Our maximum value of 50.3% is very well within this range. It has to be noted that our way of continuously pulsing does not allow to measure steady-state inactivation as it does a double-pulse protocol often used in whole-cell experiments. The voltage dependence of inactivation (Fig. 2) has a steepness of 7 mV per e-fold change of availability, which is consistent with single-channel data from guinea-pig [7, 23, 31]. Compared to Ochi and Kawashima [31]the midpoint seems shifted negative by almost 30 mV. This might be explained by the fact that they worked at a higher temperature of 30–35°C.

Our activation curve is not directly comparable to previous animal studies. Either the activation curve had been normalized to one [23], the exact membrane potential was unknown [23]or calcium was used as the charge carrier [38]. However, we were primarily interested in finding a voltage at which the maximum availability is reached and at which openings and closures can be still resolved. This was the case at a test potential of +20 mV. Interestingly, the open probability seems to increase over a broader voltage range than the availability [5, 6, 37]. This can be also seen from Fig. 1A, where popen continuously increases at positive test potentials.

Third, the conductance was in the range of 17 pS which is in clear excess of the 7–8 pS typical for T-type channels [46]. The conductance we found falls behind an often-cited value of {approx}25 pS for L-type channels (e.g. [16]) because of two reasons. The concentration of 70 mM of a divalent is a non-saturation concentration [6, 49], and most previous studies were performed with 100 or 110 mM Ba2+. For instance, Pelzer et al. [33], using guinea-pig myocytes, reported a unitary conductance of {approx}20 pS under control conditions with 90 mM Ba2+ in the pipette. Furthermore, single-channel conductance measurements have been often performed in the presence of calcium-channel agonists which facilitate resolving full-amplitude openings, especially at more negative test potentials. In fact, we were able to find higher conductance values in the presence of FPL64176. Therefore, our conductance measurements are just within the expected range for L-type channels [1].

Fourth, the sensitivity towards calcium-channel agonists like Bay K8644 or the benzoylpyrrole FPL64176 [35]is a unique feature of L-type calcium channels.

Now we consider the detailed kinetic properties of the channels described. After establishing a standard voltage protocol (150 ms steps from –100 to +20 mV, applied every 0.6 s), we were able to collect sufficient data from individual channels to evaluate both the rapid and slow gating parameters.

The fast gating process, i.e. the kinetics of openings and closures within single sweeps, is characterized by an open time distribution that can be adequately fit by a simple exponential, and by a double-exponential closed time distribution, respectively. This is in agreement with many animal experiments (e.g. guinea-pig ventricle [6, 7]; neonatal rat heart [5]; bovine chromaffin cells [9]) as well as with the findings of Jahnel et al. [20]for human atrial L-type channels and conforms to the C{leftrightarrow}C{leftrightarrow}O gating paradigm commonly used to describe L-type channel kinetics. We could not detect clear-cut evidence for modal gating, although occasionally sweeps with unusually long openings were observed in the absence of calcium agonist (e.g. Fig. 3, penultimate sweep). It may be argued, however, that the analytical strategies previously developed to identify modal gating in guinea-pig myocardium (popen histograms and to, max histograms; see [15, 48, 50]) are inadequate to see such phenomena in human channels under basal conditions. This matter, however, requires further study, involving the analysis of cAMP-dependent phosphorylation [48, 50]and the influence of the nature of the charge carrier [19]. For quantitative comparison of the determined parameters (Table 2) to those from animal studies, the experimental conditions, especially test potential, charge carrier and temperature, have to be considered. Identical conditions are found in several single-channel studies on guinea-pig ventricular myocytes [26, 27, 48]and comparable conditions were used by Hirano et al. [17]for guinea-pig and by Chen et al. [8]for rat. Table 2 shows that our results are very well within the range of what is found in these investigations (Table 2).

The slow gating process has been identified based on the non-random appearance of sweeps containing openings (‘active’) and no openings (‘blank’). Animal studies reveal that runs of consecutively active and blank sweeps are significantly clustered, indicated by the high (≥2) Z-value derived from runs analysis [7, 18]. Histograms allow an estimation of the underlying lifetime of an available and unavailable state, which both greatly exceed the duration of a pulse interval [31]. Qualitatively and quantitatively, we confirm these findings in the human channels. The active and blank run lifetime found here is in agreement with results from guinea-pig ventricular myocytes, elicited under the same experimental conditions [14, 48]. Wiechen et al. [48], for example, measured a time constant for the active run length of about 1.2 s and a mean blank run lifetime of about 3.1 s for the various control groups. Herzig et al. [14]determined a mean blank run lifetime of 5.0±1.2 s. The second component found in some of our blank run histograms is a hint for incomplete recovery from inactivation at 1.66 Hz [14, 48]. It will be interesting to see whether cAMP-dependent phosphorylation underlies these phenomena and affects the parameters in a manner previously seen in guinea-pig channels [14, 30, 31, 48].

Finally, we want to discuss our data in terms of reproducibility. Some of the determined parameters show a rather large standard error, especially the ensemble peak current, the inactivation and the open probability (Table 2). Such scatter can be found as well with animal data (e.g. see [26, 27, 45, 48]) and seems not to be specific for calcium channels from human failing hearts. When taking into account all the differences in patient characteristics, time from explantation to cell isolation, and time from cell isolation to the respective successful recordings, it is remarkable that the standard errors are in a range which is comparable to animal data without such variables. It is of course not possible to correlate the respective individual results with the patient's cause of heart failure or his medication.

In summary, we were able to determine the detailed single-channel properties of human ventricular L-type calcium channels with an interindividual scatter that is completely in the range of what is found in studies on healthy animal cardiomyocytes. The fast and slow gating characteristics are in full qualitative and the numerical values in gross quantitative agreement with data from other species. To determine whether heart failure, as present in all hearts used in this study, has any quantitative influence on the number, function and modulation of calcium channels requires further studies. Such experiments will be feasible and they are necessary, given the abnormalities in cAMP-dependent phosphorylation and dephosphorylation reactions found in heart failure (e.g. [12, 28, 41]).

Time for primary review 38 days.


   Acknowledgements
 
This study was supported by the DFG (He 1578 6-1). The technical help of Mrs. Elke Schröder and Mrs. Elke Hippauf is gratefully acknowledged. The scientific and logistic cooperation with Prof. D. Beuckelmann, Prof. M. Böhm, and Dr. R.H.G. Schwinger (Department of Cardiology, University of Cologne) is gratefully appreciated.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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