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(Circulation. 1998;98:969-976.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Increased Availability and Open Probability of Single L-Type Calcium Channels From Failing Compared With Nonfailing Human Ventricle

Frank Schröder, MD; Renate Handrock, PhD; Dirk J. Beuckelmann, MD; Stephan Hirt, MD; Roger Hullin, MD; Leo Priebe, MD; Robert H. G. Schwinger, MD; Joachim Weil, MD; ; Stefan Herzig, MD

From the Departments of Pharmacology (F.S., R.H., S. Herzig) and Cardiology (D.J.B., L.P., R.H.G.S.), University of Cologne; the Department of Cardiothoracic Surgery, University of Kiel (S. Hirt); the Department of Cardiology, Ludwig-Maximilians-University, Munich (R.H.); and the Department of Pharmacology, University of Hamburg (J.W.), Germany.

Correspondence to Stefan Herzig, MD, Department of Pharmacology, University of Cologne, Gleueler Straße 24, 50931 Cologne, Germany. E-mail stefan.herzig{at}uni-koeln.de

	Abstract

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Background—The role of the L-type calcium channel in human heart failure is unclear, on the basis of previous whole-cell recordings.

Methods and Results—We investigated the properties of L-type calcium channels in left ventricular myocytes isolated from nonfailing donor hearts (n=16 cells) or failing hearts of transplant recipients with dilated (n=9) or ischemic (n=7) cardiomyopathy. The single-channel recording technique was used (70 mmol/L Ba²⁺). Peak average currents were significantly enhanced in heart failure (38.2±9.3 fA) versus nonfailing control hearts (13.2±4.5 fA, P=0.02) because of an elevation of channel availability (55.9±6.7% versus 26.4±5.3%, P=0.001) and open probability within active sweeps (7.36±1.51% versus 3.18±1.33%, P=0.04). These differences closely resembled the effects of a cAMP-dependent stimulation with 8-Br-cAMP (n=11). Kinetic analysis of the slow gating shows that channels from failing hearts remain available for a longer time, suggesting a defect in the dephosphorylation. Indeed, the phosphatase inhibitor okadaic acid was unable to stimulate channel activity in myocytes from failing hearts (n=5). Expression of calcium channel subunits was measured by Northern blot analysis. Expression of _1C- and ß-subunits was unaltered. Whole-cell current measurements did not reveal an increase of current density in heart failure.

Conclusions—Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.

Key Words: calcium channels • heart failure • myocytes

	Introduction

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Cardiac excitation-contraction coupling depends on the function of L-type calcium channels. One may speculate that calcium channel dysfunction may be involved in the pathophysiology of heart failure. Numerous studies have addressed the issue in animal models^{1 2 3 4 5 6 7 8 9 10 11 12} or patient material,^{13 14 15 16 17} mostly by measuring dihydropyridine binding or the whole-cell calcium current density. The findings are inconsistent, with increases,^{1 2} decreases,^{3 4 5 6 7 8 13 17} or no change^{9 10 11 14 15 16} reported. This may be related to species differences, the model or severity¹² of failure, or the assay used.² Importantly, studies on human material revealed a slight reduction in calcium channel mRNA expression and dihydropyridine binding sites¹³ but an unchanged whole-cell current under both basal^{15 16} and forskolin-stimulated conditions.¹⁵

It is premature to conclude that calcium channel alterations are irrelevant to human heart failure. The whole-cell current I is a function of both the number of functional channels N and their individual properties i (single-channel current amplitude), the open probability (p_open, fraction of time spent in the open state during active sweeps), and the availability (f_active, fraction of active sweeps per number of test pulses), where I=Nxixp_openxf_active. Therefore, any incongruence between N and I could in theory be accounted for by alterations of i, p_open, or f_active. Because the latter 2 parameters are known to be modulated physiologically by cAMP-dependent phosphorylation,^{18 19 20 21 22} it should be interesting to measure them under the conditions of heart failure. We recently demonstrated²³ that single L-type channel recording is possible in human ventricular myocytes. Here, we report that heart failure markedly increases single-channel current because of increased open probability and availability.

	Methods

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Preparation of Cardiomyocytes
Ventricular myocytes were prepared from failing or nonfailing hearts. Failing hearts were obtained from patients with end-stage heart failure caused by ischemic or dilated cardiomyopathy (IC, DC) who were undergoing transplantation. Nonfailing hearts were from organ donors who had died of noncardiac causes, whose hearts could not be transplanted for various technical reasons. According to the available patient data, there were no significant differences between the groups regarding age (failing, 57.5±1.2 years; nonfailing, 51.5±4.2 years) or sex (66% male). The medication of transplant recipients regularly included diuretics, ACE inhibitors, and digitalis glycosides. Some of the organ donors received intravenous catecholamines (dopamine, norepinephrine) until surgery.

The tissue was placed into ice-cold cardioplegic solution and shipped to the laboratory within 18 hours. It was placed in preoxygenated solution A (4°C) composed of (mmol/L) NaCl 100, KCl 10, MgSO₄ 5, dextrose 20, taurine 50, and MOPS 5 (pH 7.4). After removal of fat and connective tissue, slices 2x2x0.5 mm were cut from the free left ventricle and enzymatically digested²³ in 10 mL of solution A containing collagenase (1.5 mg/mL, type CLS 1, Worthington Biochemical Corp), trypsin (1 mg/mL, type III, Sigma Chemical Co), and BSA (10 mg/mL, Sigma) at 37°C for 40 minutes. A second incubation (30 to 90 minutes, depending on the cell yield checked at 10-minute intervals) in the presence of collagenase (0.5 mg/mL) and BSA (1 mg/mL) followed. After gravity settling (in solution A, 15 minutes), cells were placed in solution B containing (mmol/L) potassium glutamate 50, KCl 40, KH₂PO₄ 20, taurine 20, KOH 20, MgCl₂ 3, HEPES 10, EGTA 5, dextrose 10 (pH 7.4, 22°C), disaggregated, and stored at 4°C (1 hour) before use. Then, the cell suspension was incubated (30 to 180 minutes, 22°C) with 10 µmol/L BAPTA-acetoxymethyl ester to buffer intracellular divalent cations.

Electrophysiological Measurement
Cells were placed in disposable perfusion chambers (3 mL) containing a bath solution of (mmol/L) NaCl 135, KCl 4, MgCl₂ 1, HEPES 10, CaCl₂ 2, dextrose 10 (pH 7.4 with NaOH, 21°C to 23°C). Pipettes (borosilicate glass, 7 to 10 M) were filled with (mmol/L) 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. Barium currents were elicited by depolarizing test pulses of 150 ms at 1.66 Hz (see References 21, 23, and 24^{21 23 24} ), recorded at 10 kHz, and filtered at 2 kHz (-3 dB, 4-pole Bessel) with an Axopatch 200 A amplifier (Axon Instruments). Command pulses were 120 mV in amplitude (eg, from -100 to +20 mV or from -40 to +80 mV, depending on the resting potential of the cell), with absolute values adjusted to yield single-channel amplitudes of -0.7 nA. This corresponds to a test potential of +20 mV across the patch membrane, where channel availability is maximal (see Reference 23²³ , Figure 2). Only the experiments without a shift in single-channel current amplitude (gauged by amplitude histograms) were evaluated. PClamp software (version 6.0, Axon Instruments) was used for acquisition and analysis. 8-Br-cAMP (from Sigma, 0.1 mol/L stock in DMSO) and okadaic acid (NH₄⁺ salt, from Calbiochem, 0.1 mmol/L stock in DMSO) were added to the bath as a 30-µL bolus. The final drug concentrations depended on the exact amount of the bath volume, determined after the experiment. The final concentrations amounted to 0.84±0.04 mmol/L (from 0.6 to 1.1 mmol/L) 8-Br-cAMP and 0.86±0.07 µmol/L (0.7 to 1.1 µmol/L) okadaic acid.

View larger version (30K): [in this window] [in a new window]   Figure 2. Open time (top) and closed time (bottom) histograms of 2 experiments depicted in Figure 1 (left, channel from nonfailing heart; right, channel from failing heart). Curves were generated with a maximum-likelihood estimate for simple (open times) or double exponential (closed times). Time constants amounted to open=0.64 ms, closed,fast=0.51 ms, and closed,slow=25.8 ms for channel from nonfailing heart and open=0.54 ms, closed,fast=0.34 ms, and closed, slow=11.4 ms for channel from failing myocardium.

Data Analysis
Experiments were analyzed whenever the channel activity persisted for at least 72 seconds (120 sweeps) both under control conditions and after exposure to drug. Linear leak and capacity currents were digitally subtracted. The availability (fraction of sweeps containing at least 1 channel opening), the open probability (p_open, defined as the relative occupancy of the open state during active sweeps), and the peak ensemble average current (i_peak, obtained after optical or mathematical smoothing) were analyzed from single-channel and multichannel patches. In the latter case, they were corrected for n, the number of channels in the patch. n was the maximum current amplitude observed divided by the unitary current. Peak current was corrected by division through n. The availability was corrected by the square root method: (1-availability_corrected) is the nth root of (1-availability_uncorrected). The corrected p_open was calculated on the basis of the corrected number of active sweeps, ie, total open time divided by (nxavailability_correctedxnumber of test pulses). Openings and closures were identified by the half-height criterion. Closed-time and first-latency analyses were carried out in 1-channel patches only. First latency was determined by averaging the waiting times between the beginning of the test pulse and the first opening (if present). Open-time and closed-time histograms were fitted with a maximum-likelihood estimate (PStat software, Axon Instruments) of log-binned data. Slow gating was analyzed in experiments (with only 1 channel in the patch) that contained at least 300 sweeps. The sweep histograms and probability plots were fitted by least-squares methods. Two-tailed t tests were used for statistical comparisons, with either the unpaired or paired format as appropriate. Values are given as mean±SEM.

Whole-Cell Experiments
Cells were isolated as described.¹⁶ The bath solution was (mmol/L) choline chloride 130, HEPES 25, dextrose 22, 4-aminopyridine 4, CaCl₂ 2, and MgCl₂ 1.1, pH 7.4 (with TEA-OH). Peak inward calcium currents were measured (similar hardware to that for single channels) at steady state, with 200-ms steps applied every 2 seconds from a holding potential of -80 to +10 mV (peak of current-voltage relation). The recording pipette contained (mmol/L) CsCl 140, HEPES 25, and fura-2 0.05, pH 7.2 (with TEA-OH). Current density was calculated by dividing peak current through cell capacitance.

Northern Blots
Preparation of poly(A) mRNA and quantification of transcripts for the calcium channel _1C- and ß-subunits by Northern blot analysis were carried out as previously described.²⁵ Samples were taken from the left ventricle of the same hearts from which electrophysiological data were obtained. Calsequestrin expression was used for normalization of the RNA yield, because transcription of this gene is unaltered in heart failure.¹³ _1C-mRNA was detected by hybridization with a 448-base cRNA complementary to a region of the human cardiac _1C, which includes the IV S6 transmembrane segment. ß-Subunit mRNA was identified by hybridization with a 411-base cRNA coding for a central core region of the human ß-subunit. Cardiac calsequestrin expression was quantified by hybridization with a 190-base cRNA coding for the carboxy terminus of calsequestrin. Hybridization reactions for all transcripts were done subsequently on the same gels at 42°C.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Single-channel activity of L-type calcium channels is markedly enhanced in failing myocardium compared with nonfailing controls. This is illustrated by original recordings from 2 experiments (Figure 1) and by the corresponding open-time and closed-time histograms (Figure 2). The increase in ensemble average current (Figure 1, bottom traces) is due both to an increased availability and to an increased open probability. The latter effect is caused predominantly by shorter closed times, as seen in the histogram analysis (Figure 2). These findings were statistically significant (Figure 3) and independent of the cause of heart failure. Table 1 presents the details. It shows that the higher open probability of channels from failing hearts is due to 3 reasons: a shorter first latency, a longer mean open time, and a shorter closed time (faster time constant of the slow component). The unitary current amplitude i is similar between the 2 groups, which is trivial, because we adjusted our pulse protocol according to this parameter (see Methods). Importantly, single-channel conductance, obtained by measuring the amplitudes of fully resolved openings at 2 different test potentials, is identical between channels from nonfailing (16.7±3.2 pS, n=6) and failing (16.8±1.7 pS, n=11) myocardium. This value also matches our previous findings (16.6±1.2 pS, n=6) in channels from failing myocardium (see Reference 23²³ for detailed discussion of absolute value) in a potassium-rich solution, for which the membrane potential is exactly known.

View larger version (40K): [in this window] [in a new window]   Figure 1. Comparison of single L-type calcium channels from nonfailing organ donor heart (left) and terminally failing heart (right). Top row, Pulse protocol (150-ms steps, amplitude 120 mV, applied every 600 ms). Applied voltages were from -70 to +50 mV (left) and from -100 to +20 mV (right). Middle, 20 consecutive sweeps for each channel. Bottom rows, Average current of all 240 (left) or 300 (right) sweeps of entire ensembles. Scale bars=20 ms and 2 pA (unitary current traces) or 17.5 fA (ensemble averages).

View larger version (27K): [in this window] [in a new window]   Figure 3. Statistical analysis of channel behavior regarding peak current ipeak, open probability popen, and availability of channels recorded from cells isolated from nonfailing donor hearts (open bars, n=16 channels from 9 hearts) or failing hearts (hatched bars, all: n=16 experiments from 10 failing hearts, composed of n=9 experiments from 5 hearts with DC and n=7 experiments from 5 hearts with IC). Comparison between nonfailing and all failing hearts (first 2 bars in each graph) was done by 2-tailed t test (*P<0.05). To check for possible differences depending on cause of failure, ANOVA was performed on nonfailing, DC, and IC data (*P<0.05 by 1-way ANOVA). In post hoc tests, significant results in ANOVA were due to difference between nonfailing and IC groups (P<0.05 after Bonferroni correction, not depicted). There were no significant differences between DC and IC.

View this table: [in this window] [in a new window]   Table 1. Comparison of Single-Channel Properties of L-Type Channels From Nonfailing and Failing Human Ventricular Myocytes

The above-mentioned profile of the enhanced activity of channels from failing ventricles resembles the pattern of cAMP-dependent stimulation of cardiac L-type channels known from animal experiments.^{18 19 20 21 22} It was therefore of interest to compare this profile with the effects of a cAMP analogue. Our first preliminary attempts to modulate human cardiac L-type channels by 8-Br-cAMP were fruitless: in none of 5 technically successful bath applications of the drug did current increase under depolarizing conditions (see References 20 and 23^{20 23} ); therefore, the present study used a physiological bath solution. This change in condition allowed us to obtain a stimulation by 8-Br-cAMP, as shown by the time course of such an experiment (Figure 4). Here, both the availability (density of bars) and the open probability (bar height) were markedly elevated in a channel from a nonfailing heart. This is also seen in Figure 5, which shows representative traces from the same experiment and the average currents from the entire ensembles. When the results from all technically successful drug applications were combined (n=3 patches from 3 nonfailing hearts, n=9 patches from 8 failing hearts), the peak current was elevated (from 39.6±12.9 to 63.8±19.4 fA, P<0.05) because of both effects, but only the increase in open probability (from 7.84±2.01% to 9.72±2.56%) and not the availability (from 43.8±8.7% to 57.3±7.5%) reached statistical significance in a 2-tailed paired analysis. This was rather unexpected, given the higher sensitivity²² and the lower variability (eg, see Reference 24²⁴ ) of phosphorylation-dependent effects on availability compared with open probability in animal experiments. Inspection of the data from all individual experiments (Figure 6) and separate analysis of results from nonfailing versus failing tissue (Table 2), however, give a clue for this phenomenon. Whereas the experiments with cells from nonfailing hearts show a strong increase in current (from 14.1±12.4 to 40.9±38.6 fA, n=3, P=NS), channels from failing myocardium start off at a very high baseline (at 48.0±16.2 fA, n=9), as expected (see Table 1), and peak current can be raised only slightly, to 71.4±24.2 fA (P=0.07, 2-sided paired t test). In fact, the availability is sometimes close to its theoretical maximum, and it is clear that these channels cannot show any further increase after 8-Br-cAMP. In summary, the same picture emerges when channel activity from nonfailing versus failing hearts on the one hand and the effect of cAMP-dependent phosphorylation on the other hand are compared: the first latency is shorter, the open time higher, the closed times lower, the open probability higher, and the peak average current higher both in cells from failing hearts and after 8-Br-cAMP. This raises the idea that a phosphorylation-related mechanism is responsible for the elevated activity of channels from failing heart.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of 8-Br-cAMP (0.75 mmol/L) on activity of single channel in ventricular myocyte from nonfailing myocardium. Open probability (popen) increases after drug addition.

View larger version (42K): [in this window] [in a new window]   Figure 5. Single consecutive traces from experiment in Figure 4, illustrating mechanism of cAMP-dependent stimulation. Left, Before 8-Br-cAMP; right, after 8-Br-cAMP. Pulse protocol (top row) consisted of 150-ms steps from -100 to +20 mV throughout experiment. Ensemble averages (bottom rows) were calculated from all 540 sweeps before (left) and 600 sweeps after drug addition. Scale bars=20 ms and 2 pA (unitary current traces) or 46 fA (ensemble averages).

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of 8-Br-cAMP in all technically successful experiments. Channels from failing hearts () are characterized by a higher control value before drug and a smaller drug effect than channels from nonfailing hearts (). This is especially true for channel availability.

View this table: [in this window] [in a new window]   Table 2. Effect of 8-Br-cAMP on Single-Channel Behavior

To examine this idea further, we analyzed the kinetic properties of the slow gating process, ie, the movement of channels between an "available" and an "unavailable" state, as evidenced by the nonrandom occurrence (clustering) of active and blank sweeps.^{19 21 23} First, the lifetime of the available state was estimated by sweep histogram analysis, in which the distribution of "runs" (ie, series of continuously active sweeps or continuously blank sweeps) is plotted against time. For the duration of active runs, we obtained the rate constants of monoexponential fits (not shown) from the long-lived recordings with 1 channel in the patch. This value was significantly (P=0.006) decreased in channels from failing myocardium (0.562±0.108 s^-1, n=7) compared with nonfailing myocardium (1.757±0.328 s^-1, n=8). Blank run duration was biexponential in 4 of 8 cells from nonfailing tissue, consistent with previous animal data.²¹ Channels from failing myocardium, in contrast, revealed a monophasic blank run distribution. This is also seen in the probability plots averaged from all these experiments (Figure 7). With the plain sweep histogram analysis used so far, there are 2 methodological limitations. First, silent transitions between states occurring during the test pulse interval are missed and may cause numerical errors in the lifetime estimate. Second, the existence of a short-lived blank state (obviously present in nonfailing and presumably also in failing myocardium) will cause a systematic error (underestimation) of the true lifetime of the "phosphorylated" available state (see Reference 24²⁴ ). To circumvent these problems, we used a discrete-time Markov model.²¹ This model consists of an available state, A, linked to 2 nonavailable states, L and S (for long- and short-lived), and these transitions are taken to be mediated by a phosphorylation- and a non–phosphorylation-linked event, respectively. The resulting system SAL is thus described by 4 rate constants. All data sets in Figure 7 were fitted simultaneously. To reduce the degree of freedom for the fit, we assumed identical SA transitions for nonfailing and failing myocardium (it would be impossible to determine these rates in the failing data alone). The curves shown in Figure 7 correspond to the best-fit rate constants, in which exit from the phosphorylated state (AL) occurs at 0.137 s^-1 in channels from failing myocardium and at 0.798 s^-1 in channels from nonfailing myocardium. The reverse phosphorylation reaction LA is not too different between the 2 groups (failing, 0.318 s^-1; nonfailing, 0.269 s^-1). For both data sets, the fast rate constants AS and SA were 0.633 s^-1 and 2.08 s^-1, respectively.

View larger version (24K): [in this window] [in a new window]   Figure 7. Analysis of slow gating of channels from nonfailing (left) and failing (right) hearts. Only those experiments with 1 single channel and a long recording time (300 sweeps) were used (n=8 for nonfailing, n=7 for failing). Length of runs of consecutive active (top) or blank (bottom) sweeps was counted; probability of active or blank to be at least x seconds long was calculated and then averaged. Calculated curves were generated with a discrete-time Markov model, as explained in detail in text. Data show that duration of active state is prolonged in channels from failing hearts, whereas overall duration of blank states is shortened.

This finding suggests that the enhanced availability, indicative of a higher steady-state level of phosphorylation in channels from failing myocardium, is primarily due to an impaired dephosphorylation reaction, with little or no change in the phosphorylation rate constant. Because channel availability is controlled primarily by a type 1 protein phosphatase in animals^{21 24} and because this phosphatase is itself controlled by cAMP-dependent phosphorylation,^{21 26} we investigated whether this regulation is still present in channels from failing myocardium. Interestingly, the active run durations remained unaffected by 8-Br-cAMP (rate constants changed from 0.618±0.168 s^-1 to 0.652±0.215 s^-1, P=NS) in those 4 single-channel experiments in cells from failing hearts in which the availability was elevated by the drug. Kinetically, the stimulation was due to a decrease of the blank run lifetime (because the rate constants tended to increase from 0.735±0.273 s^-1 to 1.055±0.212 s^-1, P=0.08, n=4), suggesting a pure effect on the phosphorylation reaction. In contrast, the 2 single-channel patches from nonfailing myocardium revealed the known²¹ dual response, ie, a >2.5-fold change of both constants, namely, a decrease in the blank run lifetime and an increase in the active run lifetime. These findings suggest that a type 1 protein phosphatase, which normally controls L-type calcium channel availability, is downregulated in heart failure.

To further test the role of protein phosphatases for channel activity, we applied the membrane-permeant phosphatase inhibitor okadaic acid to n=5 patches (1 single-channel, 4 multichannel) from failing myocardium. No significant stimulatory effect was found for peak current (from 35.3±14.6 to 40.8±19.3 fA), open probability (from 8.5±5.1% to 9.2±5.3%), or availability (from 45.8±8.9% to 47.5±10.5%). This is in striking contrast to the profound stimulation we found previously in guinea pig myocardium under identical conditions²⁴ and to the effect of okadaic acid on a single channel recorded from a nonfailing heart (peak current was raised from 4.5 to 16.5 fA, open probability from 0.8 to 3.1%, and availability from 32% to 41%). These findings further support the idea that a downregulation of channel dephosphorylation is the reason why channels from failing myocardium are more active.

To examine the idea of whether the number of functional channels is also affected by heart failure, we subjected tissue samples of the same hearts as studied at the single-channel level to a whole-cell study and to a Northern blot analysis. In whole-cell recordings, the cell capacitance (nonfailing, 196±26 pF, n=4 cells from 3 hearts; failing, 192±26 pF, n=6 cells from 4 hearts) and the current density (nonfailing, 5.1±1.5 pA/pF; failing, 2.4±0.6 pA/pF) were not significantly altered. Expression of mRNA for the calcium channel subunits _1C and ß was measured in 18 sufficiently large tissue specimens from 13 hearts (5 nonfailing and 8 failing), and successful RNA preparation and subsequent hybridization reactions with the probes for _1C- and ß-subunits took place in 13 cases (4 specimens from 4 different nonfailing hearts and 9 specimens from 7 different failing hearts). There were only subtle, insignificant changes of expression of the _1C- and ß-subunits (Figure 8). However, there was a large scatter, especially for the values from nonfailing hearts. The ratio of ß-subunit mRNA over _1C-subunit mRNA was significantly reduced in heart failure (nonfailing, 8.10±3.20 compared with 3.16±0.59 in failing hearts), possibly indicating an altered subunit composition. In summary, these data indicate that the profound changes seen at the single-channel level are not reflected in similar alterations of overall current density or in clear reciprocal changes of mRNA expression.

View larger version (34K): [in this window] [in a new window]   Figure 8. Northern blot analysis of human cardiac tissue samples. Left, Radiolabeled antisense probes detected transcripts of 8-kb size for 1C-subunit, 5.6-kb for ß-subunit, and 2.6-kb for calsequestrin in nonfailing and failing myocardium. Each lane contained 10 µg of RNA. Exposure time for autoradiography was 120 hours (1C), 72 hours (ß), and 16 hours (calsequestrin). Right, Hybridization signals were analyzed densitometrically and normalized for calsequestrin signal of respective sample. No significant changes were seen.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The main finding of our study is a dramatically increased activity of L-type calcium channels in human heart failure. At first glance, this effect is in apparent conflict with data from the literature suggesting unchanged or depressed calcium currents, at least in the majority of studies. It must be remembered, however, that none of the previous investigations, in animals or humans, have addressed the question at the single-channel level. As delineated in the introduction, the whole-cell current I is defined as I = Nxixp_openxf_active. An unchanged value for I may reflect no change at all or any sort of reciprocal alterations in the other terms. We find no evidence for a change in the permeation properties, which determine the size of the single-channel amplitude. However, both p_open and f_active are increased. We were able to confirm the previous finding of unaltered^{15 16} or perhaps slightly reduced¹⁷ whole-cell current density I in heart failure. This means that the number of functional channels, N, must be largely reduced in heart failure, ie, by a factor of 2- or 3-fold. Previous Northern blot experiments as well as some dihydropyridine binding studies indeed showed a reduced channel expression in human heart failure.¹³ We were unable to confirm a reduced expression at least of _1C-subunit mRNA, possibly because of the small number of samples and the large scatter. In future studies, the number of functional channels might be addressed by simultaneous measurements of whole-cell and single-channel currents.

Considering the mechanism of the elevated activity of channels from failing hearts, the similarity with the effects of a cAMP derivative suggests a phosphorylation-dependent mechanism. Although chronic effects of the standard medical treatment with diuretics, ACE inhibitors, and digitalis cannot be ruled out in this study, we do not believe that acute ß-adrenergic effects arising in vivo from the noradrenergic tone in heart failure were conserved in our experiments. The tissue and cells had been washed 12 times, the time between explantation and our measurements ranged from 4 to >24 hours, and cells from nonfailing hearts of donors treated with intravenous catecholamines did not show such increased activity. However, it seems counterintuitive to attribute the elevated single-channel activity to an increased baseline of phosphorylation, given the presumed deficit in PKA-mediated protein phosphorylation in failing human myocardium.^{27 28} One may speculate that the channel behavior indicates, instead of increased phosphorylation state, an uncoupling from (dephosphorylated) inhibitory subunits. Both the _1C-¹⁹ and the ß-subunits³⁰ are target proteins for cAMP-dependent phosphorylation of cardiac calcium channels, and we found a reduced relative abundance of ß-subunit mRNA. Alternatively, an increased phosphorylation state of the channel may also result from a decrease in the dephosphorylation rate. Our data provide a kinetic picture at the single-channel level. Here, the results on slow gating are entirely compatible with an increased channel phosphorylation state despite unaltered kinase activity: the prolonged dwell time of the available state probably reflects a reduction in dephosphorylation rate, and a reduced type 1 protein phosphatase activity could be responsible. Accordingly, inhibition of protein phosphatases by okadaic acid was ineffective in channels from failing myocardium. Interestingly, protein phosphatase activity in tissue samples from failing human heart is increased in sarcoplasmic reticulum membrane preparations³¹ but not in homogenates from whole tissue,³² suggesting an altered subcellular distribution of phosphatases.

The alterations in the rapid gating parameters also deserve consideration: an increased open probability is the single-channel manifestation of voltage- or frequency-induced potentiation (facilitation) of the channel.^{24 33} Our findings may thus be reflected at the whole-cell level by the altered high-frequency–induced facilitation of calcium current recently reported for failing human heart.³⁴ If pertinent to physiological conditions, this type of change in single-channel gating, when associated with altered deactivation properties,²⁰ may cause proarrhythmic effects by early afterdepolarizations.³⁵

Important questions remain unresolved at present. What is the activity of calcium channels located in the T tubules and closely coupled to ryanodine receptors and EC coupling? In the cell-attached mode, only superficial channels are seen, and channels in T tubules may behave differently. This would explain the results of Gomez and coworkers,¹¹ who showed an impaired coupling between calcium currents and calcium release sparks from the sarcoplasmic reticulum in a rat model. Single-channel data under their conditions would be interesting. It is also unclear whether the increase in single-channel activity is a primary or a secondary event. Sarcoplasmic reticulum proteins are dysfunctional in heart failure (see Reference 36³⁶ ), and reciprocal regulation of calcium current by expression of ryanodine receptors has been found.³⁷ It is therefore feasible that the increased activity of superficial channels just compensates for a reduced channel expression in the T tubules. A relative lack of ß-subunit could contribute to such an altered distribution of channels.³⁸ Alternatively, a reduced calcium channel expression could also be primary, because of the increased noradrenergic tone in heart failure, as shown in cell culture.³⁹ The increased activity of remaining channels would then be a secondary compensatory phenomenon.

In summary, our findings emphasize an important role of the L-type calcium channel in the pathophysiology of human cardiac failure. Its markedly increased single-channel activity indicates that it is not an innocent bystander in heart failure.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (He 1578 6–1). We gratefully acknowledge Elke Hippauf for excellent technical help and Ursula Kreuzberg for contributing to some experiments and analyses.

Received January 29, 1998; revision received April 17, 1998; accepted April 22, 1998.

	References

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