Sodium-calcium exchange in heart: membrane currents and changes in [Ca2+]i.

Recordings have been made of changes in intracellular calcium ion concentration ([Ca2+]i) that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Guinea pig ventricular myocytes under voltage clamp were perfused internally with fura-2, a fluorescent Ca2+-indicator, and changes in [Ca2+]i and membrane current that resulted from Na-Ca exchange were identified through the use of various organic channel blockers and impermeant ions. Depolarization of cells elicited slow increases in [Ca2+]i, with the maximum increase depending on internal [Na+], external [Ca2+], and membrane voltage. Repolarization was associated with net Ca2+ efflux and a decline in the inward current that developed instantaneously upon repolarization. The relation between [Ca2+]i and current was linear, and the slope was made steeper by hyperpolarization.

Characterization of the hyperpolarization-activated inward current in isolated human atrial myocytes.

OBJECTIVE: The hyperpolarization-activated inward current (I(f)) has been discussed to contribute to arrhythmias in rat hypertrophied and human failing ventricular myocardium. Cat atrial myocytes were found to exhibit variable size of I(f). In the present study, we evaluate characteristics of I(f) in human atrial myocardium and investigate if human atria might exhibit any electrophysiological heterogeneity in the diastolic voltage range. METHODS AND RESULTS: The whole-cell patch-clamp technique was used to record I(f) in isolated myocytes from 96 human right atrial appendages. I(f) was observed in 95% (ruptured-patch; 141/146) to 100% (perforated-patch; 18/18) of myocytes showing typical current properties, i.e. time- and voltage-dependence, block by [Cs+]o, permeability for K+, Na+ and Li+, and current increase with raising [K+]o. Using the perforated-patch technique Boltzmann distributions yielded an activation threshold of -60 to -70 mV and half maximal activation at -89.3 +/- 0.7 mV (n = 18). Isoproterenol (10(-6) mol/l) shifted I(f) activation by +7 mV (7/7) using the perforated-patch technique, but only inconsistently shifted I(f) activation using the ruptured-patch method (6/21). Based on the relative current size of I(f) and IK1 three cell types could be distinguished (n = 26). In myocytes with a prominent I(f), I(f) density was found to be larger (in [K+]o 25 mmol/l at -80 mV: -0.78 +/- 0.23 pApF-1; n = 7) than in cells with predominant IK1 (in [K+]o 25 mmol/l at -80 mV: -0.02 +/- 0.01 pApF-1; n = 4) (P < 0.05). CONCLUSIONS: I(f) is present in most human atrial myocytes. Many current properties are similar to those described for I(f) in mammalian pacemaker cells. The relative current size of I(f) and IK1 were found to be variable in different myocytes. Whether I(f) may favor spontaneous diastolic depolarization in individual human atrial myocytes exhibiting predominantly I(f) in vivo remains to be defined, as current size is very small under physiological [K+]o.

Characterization of a [Ca2+]i-dependent current in human atrial and ventricular cardiomyocytes in the absence of Na+ and K+.

OBJECTIVES: In situations of [Ca2+]i-overload, arrhythmias are believed to be triggered by delayed afterdepolarizations, which are generated by a transient inward current ITI. This study was designed to examine [Ca2+]i-dependent membrane currents in the absence of the Na+/Ca(2+)-exchanger as possible contributors to ITI in human cardiac cells. METHODS: The whole cell voltage clamp technique was used for electrophysiological measurements in human atrial and ventricular cardiomyocytes. [Ca2+]i-measurements were performed using the fluorescent Ca(2+)-indicator fura-2. All solutions were Na(+)-free. Voltage-independent [Ca2+]i-transients were elicited by rapid caffeine applications. RESULTS: In atrial myocytes, caffeine induced a transient membrane current in the absence of Na+ and K+. This current could be suppressed by internal EGTA (10 mM). Cl- did not contribute to this current. Experiments with different cations suggested non-selectivity for Cs+ and Li+, whereas N-methyl-D-glucamine appeared to be impermeable. Voltage ramps indicated a linear current-voltage relation in the range of +80 to -80 mV. Fluorescence measurements revealed a dissociation between the time courses of current and bulk [Ca2+]i-signal. In ventricular cardiomyocytes, caffeine failed to induce transient currents in 54 cells from 22 different patients with or without terminal heart failure. CONCLUSIONS: In human atrial cardiomyocytes, a [Ca2+]i-dependent nonspecific cation channel is expressed and may contribute to triggered arrhythmias in situations of [Ca2+]i-overload. No evidence could be found for the existence of a [Ca2+]i-dependent chloride current in atrial cells. In ventricular cells, neither a [Ca2+]i-dependent nonspecific cation channel nor a [Ca2+]i-dependent chloride channel seems to be expressed. Possible delayed afterdepolarizations in human ventricular myocardium might therefore be carried by the Na+/Ca(2+)-exchanger alone.

Intracellular calcium handling in isolated ventricular myocytes from cardiomyopathic hamsters (strain BIO 14.6) with congestive heart failure.

Intracellular [Ca2+]i handling has been shown to be altered in isolated ventricular myocytes from patients with terminal heart failure. The aim of this study was to evaluate if alterations of intracellular [Ca2+]i handling and triggering Ca2+ currents in cardiomyopathic hamsters (strain BIO 14.6) with congestive heart failure might be similar to changes found in myocytes of patients with terminal heart failure and, therefore if the hamster might serve as a model for heart failure in man. Cells were isolated from hearts of hamsters developing hereditary cardiomyopathy (CMP) (strain BIO 14.6) at 12-14 months of age with overt signs of congestive heart failure. Results were compared with age-matched, undiseased control animals (CTRL). [Ca2+]i transients and Ca2+ currents were recorded simultaneously from isolated cells under voltage clamp perfused internally with the Ca2+ indicator, Fura-2. Ca2+ current densities in myocytes from CMP hamsters were -6.6 +/- 0.6 versus -8.3 +/- 0.5 microA/cm2 (P < 0.05) in CTRL. Resting [Ca2+]i levels were not significantly different. Peak [Ca2+]i transients were significantly decreased in CMP cells (450 +/- 52 nM versus 1031 +/- 98 nM in CTRL, P < 0.05). The rate of diastolic [Ca2+]i decay was slower in cells from CMP animals (t1/2: 167 +/- 19 versus 109 +/- 16 ms; P < 0.05). A moderate negative correlation was found between cell surface area and [Ca2+]i transients (r = 0.42; P < 0.05). It is concluded that changes of intracellular [Ca2+]i handling may play an important role in altered contractility of the myocardium of hamsters with hereditary cardiomyopathy in the late stage of congestive heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium sparks in human ventricular cardiomyocytes from patients with terminal heart failure.

Cardiomyocytes from terminally failing hearts display significant abnormalities in e-c-coupling, contractility and intracellular Ca(2+) handling. This study is the first to demonstrate the influence of end-stage heart failure on specific properties of Ca(2+) sparks in human ventricular cardiomyocytes. We investigated the frequency and characteristics of spontaneously arising Ca(2+) sparks in single isolated human myocytes from terminally failing (HF) and non-failing (NF) control myocardium by using the Ca(2+) indicator Fluo-3. The Ca(2+) sparks were recorded by line-scan images along the longitudinal axis of the myocytes at a frequency of 250Hz. After loading the sarcoplasmic reticulum (SR) with Ca(2+) by repetitive field stimulation (10 pulses at 1Hz) the frequency of the Ca(2+) sparks immediately after stimulation (t = 0s) was reduced significantly in HF compared to NF (4.15 +/- 0.42 for NF vs. 2.81 +/- 0.20 for HF sparks s(-1), P = 0.05). This difference was present constantly in line-scan recordings up to 15s duration (t = 15s: 2.75 +/- 0.65 for NF vs. 1.36 +/- 0.34 for HF sparks s(-1), P = 0.05). The relative amplitude (F/F(0)) of Ca(2+) sparks was also significantly lower in HF cardiomyocytes (1.33 +/- 0.015 NF vs. 1.19 +/- 0.003 HF, t = 0s) and during subsequent recordings of 15s. Significant differences between HF and NF were also present in calculations of specific spark properties. The time to peak was estimated at 25.75 +/-0.88ms in HF and 18.68 +/- 0.45ms in NF cardiomyocytes (P = 0.05). Half-time of decay was 66.48 +/- 1.89ms (HF) vs. 44.15 +/- 1.65ms (NF, P < 0.05), and the full width at half-maximum (FWHM) was 3.99 +/- 0.06 microm (HF) vs. 3.5 +/- 0.07 microm (NF, P < 0.05). These data support the hypothesis that even in the absence of cardiac disease, Ca(2+) sparks from human cardiomyocytes differ from previous results of animal studies with respect to the time-to-peak, half-time of decay and FWHM. The role of elevated external Ca(2+) in HF was studied by recording Ca(2+) sparks in HF cardiomyocytes with 10mmol external Ca(2+) concentration. Under these conditions, the average spark amplitude was increased from 1.19 +/- 0.003 (F/F(0), 2mmol Ca(2+)) to 1.26 +/- 0.01 (F/F(0), 10mmol Ca(2+)). We conclude that human heart failure causes distinct changes in Ca(2+) spark frequency and characteristics comparable to results established in animal models of heart failure. A reduced Ca(2+) load of the SR alone is unlikely to account for the observed differences between HF and NF and additional alterations in intracellular Ca(2+) release mechanisms must be postulated.

Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum.

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the beta1-adrenoreceptor and Galphai, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga-ohm seals could be obtained and patches could be excised in a way that single-channel currents could be measured, although these are not completely identified.

Modulation of the hyperpolarization-activated inward current (If) by antiarrhythmic agents in isolated human atrial myocytes.

The hyperpolarization-activated inward current (If) has been discussed to contribute to arrhythmias in human atrial myocardium. If was found to be increased by beta-adrenergic stimulation. In the present study, we evaluate the modulation of If by carbachol, adenosine and by class Ic, III and IV antiarrhythmic drugs in isolated human atrial myocytes. The whole-cell patch-clamp technique was used to record If in isolated myocytes from 18 human right atrial appendages. A typical time- and voltage-dependent hyperpolarization-activated inward current could be recorded in all cells investigated (n=56). Mean current density recorded at -130 mV was -2.8+/-1.2 pApF(-1). Both adenosine and carbachol were found to directly inhibit If in human atrial myocytes by shifting the activation curves to more negative potentials. Adenosine 10(-5) mol/l shifted the potential of half-maximal activation by -5.9+/-0.4 mV from -99.4+/-0.6 mV to -105.3+/-0.4 mV (n=8; P<0.05), and carbachol 10(-5) mol/l by -5.7+/-0.5 mV from -99.2+/-0.5 mV to -104.9+/-0.6 mV (n=6; P<0.05). The concentration-response curve of adenosine calculated by a Hill function yielded a half-maximal effect of adenosine (EC50) at a concentration of 3.6+/-0.5 micromol/l, a maximal shift of -6.5+/-0.3 mV, and a Hill coefficient (h) of 2.40. We did not observe any effect of flecainide (10(-5) mol/l; n=8), sotalol (10(-5) mol/l; n=6), amiodarone (10(-5) mol/l; n=6) or verapamil (10(-5) mol/l; n=5) on If in human atrial myocytes. However, propafenone (10(-5) mol/l) was found to reversibly reduce If current size (9/13 cells) by shifting the activation curve by -5.2+/-0.4 mV (P<0.05). In human atria adenosine- and muscarinic receptor stimulation might function as endogenous protective mechanisms inhibiting the initiation of ectopic tachycardia by reducing If current size. Propafenone may be more effective in some patients with atrial tachycardias that do not respond to other class Ic, III and IV antiarrhythmic drugs. However, it has yet to be defined whether these agents suppress atrial tachycardias via an inhibition of If in vivo.

Contributions of Ca(2+)-influx via the L-type Ca(2+)-current and Ca(2+)-release from the sarcoplasmic reticulum to [Ca2+]i-transients in human myocytes.

Experiments were performed to determine the relative contributions of direct Ca(2+)-entry through the L-type Ca(2+)-current and of Ca(2+)-release from the sarcoplasmic reticulum (s.r.) to the intracellular [Ca2+]i-transient in isolated human atrial and ventricular myocytes from patients with severe heart failure and from non-failing controls. Cells were isolated from explanted hearts of patients undergoing transplantation because of severe heart failure due to dilated or ischemic cardiomyopathy or from donor hearts which could not be transplanted for technical reasons. Ca(2+)-current densities were -2.1 +/- 0.6 pA/pF in atrial cells, -4.8 +/- 0.5 pA/pF in cells from patients with heart failure and -3.2 +/- 0.5 pA/pF in non-failing controls. [Ca2+]i-transients were significantly smaller in heart failure (370 +/- 33 nM) compared to ventricular cells from non-failing hearts (760 +/- 69 nM, p < 0.05). Atrial myocytes had average [Ca2+]i-transients of 505 +/- 38 nM. After incubation in ryanodine the average [Ca2+]i-transients were not significantly different between different cell types. The results indicate that the relative contribution of Ca(2+) released from the sarcoplasmic reticulum to the [Ca2+]i-transient is significantly smaller in heart failure. The absolute contribution of the L-type Ca(2+)-current to the transient seemed to be comparable in all cell types investigated. As the [Ca2+]i-transient in the presence of ryanodine was comparable in size in all cells, changes of the intracellular [Ca2+]i-transient in heart failure are mainly due to alterations of s.r. function in these cells.

[Value of magnesium in acute myocardial infarct]. / Stellenwert von Magnesium beim akuten Myokardinfarkt.

Experiments in animal models of myocardial infarction have provided evidence that early magnesium infusion can limit the infarct size. One mechanism that has been postulated to be of importance is a protection of the cardiomyocyte against a calcium overload during or after ischemia. We had shown that in isolated human myocytes from patient with ischemic cardiomyopathy an increase of the extracellular magnesium concentration could block the L-type-calcium current in a dose dependent manner. Until recently only small, uncontrolled studies have indicated there may be a reduction of mortality due to myocardial infarction when intravenous magnesium infusion was added to standard therapy. However, two recently published randomized studies showed different results, although similar doses of magnesium were used (70-80 mmol magnesium over 24 h). The LIMIT-2-study was a double-blind, placebo controlled investigation of over 2300 patients with suspected myocardial infarction. Magnesium infusion was associated with a reduction of the 28 day mortality by 24%. The ISIS-4-study on over 50,000 patients with suspected myocardial infarction did not show any positive effect of magnesium on mortality. Major differences between both studies were differences in thrombolysis (LIMIT-2:1/3, ISIS-4: 70%). Furthermore, in LIMIT-2 magnesium infusion was started as early as possible, whereas in ISIS-4 magnesium was given after the end of thrombolytic therapy. In can be concluded that magnesium therapy in acute myocardial infarction after thrombolytic therapy is not useful. However, in patients where thrombolytic therapy is not feasable, early infusion of magnesium may be beneficial. As side effects are minor and costs are low, a therapeutic trial may be warranted, although a final decision on the effects of magnesium cannot be made.

Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells.

1. The mechanisms that control release of Ca2+ from the sarcoplasmic reticulum (SR) of guinea-pig ventricular cells were studied by observing intracellular calcium concentration ([Ca2+]i transients) and membrane currents in voltage-clamped guinea-pig ventricular myocytes perfused internally with Fura-2. 2. Sarcolemmal Ca2+ current was identified through the use of tetrodotoxin (TTX) and Ca2+ channel antagonists (verapamil) and agonists (Bay K 8644). 3. Changes in [Ca2+]i attributable to release of Ca2+ from the SR were identified through the use of ryanodine, which abolishes the ability of the SR to release Ca2+. Ryanodine-sensitive increases in [Ca2+]i could be elicited either by depolarization or by repolarization (from depolarizing pulses to relatively positive membrane potentials). 4. At appropriate voltages, it is the initial fast change in [Ca2+]i elicited by either depolarization or repolarization that is abolished by ryanodine, and is defined here as ryanodine sensitive. 5. The amplitude of the ryanodine-sensitive [Ca2+]i transient elicited by depolarization had a bell-shaped dependence on membrane potential with a maximum of about 500 nM at 10 mV, and with the upper minimum between 60 and 70 mV. Verapamil-sensitive current activated over approximately the same potential range as the [Ca2+]i transient, with a peak amplitude at 10 mV, and a reversal potential of 65 mV. 6. When a holding potential of -68 mV and TTX (30 microM) were used, the most negative pulse potential at which activation of an inward current occurred was -49 mV while changes in [Ca2+]i occurred at -43 mV. 7. Ryanodine-sensitive increases in [Ca2+]i elicited by repolarization (tail transients) were maximal for repolarization to 0 mV. Smaller changes in [Ca2+]i than maximal were elicited by repolarization to both more positive and more negative potentials than 0 mV. The peak amplitude of the verapamil-sensitive tail currents elicited by repolarization increased continuously as the membrane was repolarized to potentials more negative than 60 mV. 8. Increasing depolarizing pulse duration beyond 10-20 ms did not increase the amplitude of the [Ca2+]i transient, but prolonged it. 9. The experimental results are compared to the predictions of two theories on the mechanism of excitation-contraction coupling: Ca2+-induced release of Ca2+ (CICR), as it has been formulated from data in skinned cardiac cells, and a charge-coupled release mechanism (CCRM), as it has been formulated to explain excitation-contraction coupling in skeletal muscle. 10. Some of the results are clearly not consistent with certain features of a charge-coupled release mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca(2+)-currents and intracellular [Ca2+]i-transients in single ventricular myocytes isolated from terminally failing human myocardium.

The purpose of the present study was to test the hypothesis that steps between the excitation of the cell membrane and contraction are altered in cardiac failure. Ca(2+)-currents and [Ca2+]i-transients were measured in single ventricular myocytes isolated from explanted hearts of patients with terminal heart failure undergoing transplantation, or from donors whose organs could not be used for technical reasons. Peak Ca(2+)-current densities were unchanged, as was the current-voltage relation. However, in myocytes isolated from severely failing hearts resting [Ca2+]i-levels were elevated, peak [Ca2+]i-transients were significantly smaller, and the diastolic decline of [Ca2+]i was markedly slowed. As the trigger for the release of Ca2+ from the sarcoplasmic reticulum is unchanged and the systolic [Ca2+]i-transient is reduced, severe heart failure can be described as partial electromechanical uncoupling.

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration.

Membrane currents and changes in intracellular calcium ion concentration ([Ca2+]i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca2+-indicator, fura-2, and changes in [Ca2+]i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs+, Ni2+), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni2+-sensitive current elicited by ramp repolarization from +90 mV to -80 mV. Ramps were sufficiently rapid that little change in [Ca2+]i occurred during the ramp. The (constant) [Ca2+]i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni2+-sensitive ramp current varied linearly with 1n[( Ca2+])i. The I-V relations at different [Ca2+]i over the range -60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca2+]i had changed. The relationship between [Ca2+]i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca2+-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca2+ to the exchanger during the [Ca2+]i-transient and the effects of membrane voltage on the exchanger.

Sodium-calcium exchange in guinea-pig cardiac cells: exchange current and changes in intracellular Ca2+.

1. Membrane currents and changes in [Ca2+]i attributable to the operation of an electrogenic Na-Ca exchange mechanism were recorded in single isolated guinea-pig ventricular myocytes under voltage clamp and internal perfusion with the Ca2+ indicator Fura-2. 2. Ionic currents that interfere with the measurement of Na-Ca exchange current were blocked through the use of caesium (Cs+), verapamil and tetrodotoxin (TTX). Entry of Ca2+ through surface membrane Ca2+ channels and release of Ca2+ from sarcoplasmic reticulum were blocked with verapamil and ryanodine, respectively. 3. In the presence of the blockers listed above, depolarization to positive membrane potentials elicited slow increases in [Ca2+]i and, after an instantaneous increase, a declining outward current. Repolarization elicited a decline in [Ca2+]i and, after an instantaneous increase, a declining inward current. The changes in [Ca2+]i and a major component of the current were abolished by nickel ions (Ni2+; 5 mM). 4. The reversal potential of the current abolished by Ni2+ (Ni2+-sensitive current) was determined at different levels of [Ca2+]i by ramp repolarizations from +80 to -80 mV (1-5 mV/ms). The reversal potential of the current increased linearly with log [Ca2+]i. As a result of the foregoing and other data, the Ni2+-sensitive current was taken to be Na-Ca exchange current (INaCa). 5. The relation between INaCa and [Ca2+]i (less than 1 microM) at constant voltage over the range of -80 to +60 mV was approximately linear. No evidence of saturation could be found; small deviations from linearity at high [Ca2+]i were in the direction expected for a minor contribution from Ca2+-activated non-specific cation current (Ehara, Noma & Ono, 1988). 6. When measured at the same [Ca2+]i, the peak INaCa upon repolarization to -80 to -140 mV seemed to approach a limiting value at very negative potentials. 7. Over the range of +40 to +160 mV INaCa (measured soon after depolarization and thus at the same [Ca2+]i) increased exponentially with clamp-pulse potential. These pulses (to potentials up to +160 mV) elicited a slow rise in [Ca2+]i with the peak at the end of the pulse also increasing exponentially with pulse potential. 8. Inward membrane currents with properties similar to those described above were also recorded in association with physiological [Ca2+]i transients, when Ca2+ channels and the sarcoplasmic reticulum were not blocked. 9. Some of the results are not consistent with certain predictions of a sequential step model, or with those of a simultaneous step model in which the internal binding site for Ca2+ is saturated, or with those of a model based only on thermodynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell swelling causes the action potential duration to shorten in guinea-pig ventricular myocytes by activating IKATP.

In guinea-pig ventricular myocytes, cell swelling by incubation in hypotonic solution caused a pronounced shortening of the action potential duration (APD90: 15.5+/-14.6% compared to control; mean +/- SD) after a latency of 12 min when the intracellular ATP concentration was 2 mM. This shortening was partially reversible within 10 min after reperfusion with isotonic solution (APD90: 80. 5+/-12.1% compared to control). With 5 mM intracellular ATP in the pipette electrode, the effect of cell swelling on the action potential was significantly reduced. Incubation with 1 microM glibenclamide, a blocker of the ATP-dependent K+ current (IKATP), abolished the swelling-induced shortening of the action potential duration, whereas incubation with 0.5 mM 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), a blocker of the swelling-induced Cl- current (ICl,swell), had no effect on the action potential duration in hypotonic solution. Simultaneous measurements of membrane currents substantiate that IKATP is the current that underlies this effect. These results suggest that in the ischaemic myocardium IKATP may be partially activated by cell swelling, resulting in a shortening of the action potential duration before the intracellular ATP concentration has fallen below 2 mM.

Simulation study of cellular electric properties in heart failure.

Patients with severe heart failure are at high risk of sudden cardiac death. In the majority of these patients, sudden cardiac death is thought to be due to ventricular tachyarrhythmias. Alterations of the electric properties of single myocytes in heart failure may favor the occurrence of ventricular arrhythmias in these patients by inducing early or delayed afterdepolarizations. Mathematical models of the cellular action potential and its underlying ionic currents could help to elucidate possible arrhythmogenic mechanisms on a cellular level. In the present study, selected ionic currents based on human data are incorporated into a model of the ventricular action potential for the purpose of studying the cellular electrophysiological consequences of heart failure. Ionic currents that are not yet sufficiently characterized in human ventricular myocytes are adopted from the action potential model developed by Luo and Rudy (LR model). The main results obtained from this model are as follows: The action potential in ventricular myocytes from failing hearts is longer than in nonfailing control hearts. The major underlying mechanisms for this prolongation are the enhanced activity of the Na+-Ca2+ exchanger, the slowed diastolic decay of the [Ca2+]i transient, and the reduction of the inwardly rectifying K+ current and the Na+-K+ pump current in myocytes of failing hearts. Furthermore, the fast and slow components of the delayed rectifier K+ current (I(Kr) and I(Ks), respectively) are of utmost importance in determining repolarization of the human ventricular action potential. In contrast, the influence of the transient outward K+ current on APD is only small in both cell groups. Inhibition of I(Kr) promotes the development of early afterdepolarizations in failing, but not nonfailing, myocytes. Furthermore, spontaneous Ca2+ release from the sarcoplasmic reticulum triggers a premature action potential only in failing myocytes. This model of the ventricular action potential and its alterations in heart failure is intended to serve as a tool for investigating the effects of therapeutic interventions on the electric excitability of the human ventricular myocardium.

Characteristics of calcium-current in isolated human ventricular myocytes from patients with terminal heart failure.

The Ca(2+)-current plays a prominent role in triggering excitation-contraction coupling in the mammalian heart. It is also a target of clinically important drugs such as catecholamines or Ca(2+)-channel blockers. Until now studies of Ca(2+)-channels in human ventricular myocardium have been hampered by the fact that adequate voltage control cannot be obtained in multicellular preparations. To characterize the properties of human myocardial Ca(2+)-currents, ventricular myocytes were isolated from explanted hearts of patients with end-stage heart failure undergoing cardiac transplantation. The current-voltage relation and voltage-dependent inactivation of L-type currents were similar to those in non-diseased guinea-pig myocardium. Currents could be stimulated with isoprenaline in a dose-dependent manner. When cells were superfused with a Na(+)-free solution in the presence of Tetrodotoxin, Cs+ and Tetraethylammonium to block interfering Na+ and K(+)-currents, depolarization from a holding potential of -90 mV to -80-(-)50 mV did not elicit any time-dependent inward-current. Changing the holding potential from -90 to -45 mV did not alter the current-voltage relation. We conclude that T-type Ca(2+)-currents do not seem to make a detectable contribution to the transmembrane Ca(2+)-influx and that L-type currents in human ventricular myocytes of patients with severe heart failure have characteristics that are similar to those in other mammalian species.

[Ca2+]i in single isolated cardiac cells: a review of recent results obtained with digital imaging microscopy and fura-2.

The use of fluorescent Ca2+ indicators to observe [Ca2+]i transients in voltage-clamped single cells has many advantages over previous methods, such as the use of aequorin in multicellular preparations, for studying excitation-contraction coupling. In the studies reviewed in this article, [Ca2+]i in single isolated mammalian ventricular myocytes was observed through the use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were generally studied with the use of inverted microscopes equipped with ultraviolet epifluorescence illumination, intensified silicon intensifier target cameras (ISIT), and (or) a photomultiplier tube. Analysis of subcellular patterns of fura-2 fluorescence was performed by digital analysis of the images obtained with the ISIT camera. Variation of membrane voltage and exposure of cells to ryanodine (which was assumed to selectively block the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The main results of these studies are the following. (1) In any population of enzymatically isolated heart cells, there are (i) mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; (ii) spontaneously contracting cells, which have a relatively elevated [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous, propagating waves of high [Ca2+]i; and (iii) cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure.

Prolongation of the action potential has been postulated to be a major reason for the altered diastolic relaxation of the heart in patients with severe heart failure. To investigate the electrophysiological basis for this action potential prolongation in terminal heart failure, K+ currents were recorded in single ventricular myocytes isolated from 16 explanted hearts of patients undergoing transplantation. Results from diseased hearts were compared with ventricular cells isolated from six undiseased donor hearts. Action potential duration was significantly prolonged in cells from patients with heart failure. A delayed rectifier K+ current was hardly detectable in most cells, and if it could be recorded, it was very small in both diseased and undiseased cells. When currents were normalized for cell surface area, the average current density of the inward rectifier K+ current was significantly reduced in diseased cells when compared with normal control cells (hyperpolarization at -100 mV, -15.9 +/- 2.2 vs -9.0 +/- 1.2 microA/cm2; P < .01). In addition, a large transient outward K+ current could be recorded in human myocytes. The average current density of the time-dependent component of this transient outward K+ current was significantly reduced in heart failure (depolarization at +40 mV, 9.1 +/- 1.0 vs 5.8 +/- 0.64 microA/cm2; P < .01). Action potential prolongation in severe heart failure may partially be explained by a reduction in current densities of the inward rectifier K+ current and of the transient outward K+ current. These alterations may thereby have a significant effect on cardiac relaxation.

Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure.

A variety of outward currents exists in ventricular myocardium of different species influencing action potential duration and electrical activity. Transient outward currents have been reported in ventricular tissue of some animals but are small or absent in others. This study was conducted to investigate whether a transient outward current exists in human ventricular myocardium and to characterize its basic electrophysiological properties. Currents were recorded from enzymatically isolated human ventricular myocytes obtained from explanted hearts of 22 patients with terminal heart failure. In almost all cells studied, a transient outward current could be recorded on depolarization to between -20 and +80 mV. The size of the transient outward current was usually large enough to mask the Ca2+ current. It could be recorded under conditions in which Ca2+ influx and intracellular Ca2+ transients were suppressed. Basic current characteristics were similar to transient outward currents observed in other species. Inactivation of the transient outward current was monoexponential, with a time constant of 54.8 +/- 3.7 milliseconds at +40 mV. Half-maximal activation occurred at 16.7 +/- 1.6 mV; half-maximal steady-state inactivation occurred at -34.5 +/- 2.3 mV. Frequency-dependent reduction of peak transient outward current was 29.8 +/- 1.4% at 2 Hz compared with resting conditions. Recovery from inactivation was voltage dependent and had a biexponential time course; the faster time constant (41.0 +/- 6.5 milliseconds at -80 mV) accounted for 86.0 +/- 5.2% of total current. The transient outward current was sensitive to 4-aminopyridine (IC50, 1.15 mM). These results indicate that a large Ca(2+)-independent transient outward K+ current is present in human ventricular myocytes that might be regulated by physiological or pathological events and is a potential site for pharmacological intervention.