Adult cardiac myocytes survive and remain excitable during long-term culture on synthetic supports.

OBJECTIVE: Cardiomyocytes can be transplanted successfully into skeletal and cardiac muscle. Our goal was to determine the feasibility of grafting cardiomyocytes onto various synthetic supports to create an excitable and viable tissue for implantation. METHODS: Adult rat cardiomyocytes were cultured over an 8-week period onto different substitutes, including human glutaraldehyde-treated pericardium (n = 3), equine glutaraldehyde-treated pericardium (n = 3), polytetrafluoroethylene (n = 8), Dacron polyester (n = 16), and Vicryl polyglactin (n = 8). RESULTS: Only the cells seeded on the Dacron survived, with the synthetic fibers colonized at 8 weeks. On the other supports, the number of myocytes progressively decreased from the first week, with their density (number of cells per square millimeter) being, after 20 days, 17 +/- 2 on the polytetrafluoroethylene and 5 +/- 1 on the human or equine pericardium compared with 45 +/- 3 on the Dacron. After 8 weeks of culture on Dacron, the sarcomeric protein (sarcomeric alpha-actinin) was detected in all cells. In addition, the staining was regularly arranged and well aligned in a striated pattern. Spontaneous beating activity was obtained. Moreover, electrical stimulation of the cell preparation resulted in the generation of calcium transients, the frequency of which followed the frequency of the electrical stimulation. CONCLUSIONS: These results suggest that adult cardiac myocytes remain viable and excitable during long-term culture on a 3-dimensional Dacron support, which might constitute a new synthetic cardiac tissue.

Dilation and action potential lengthening in cardiomyopathic Syrian hamster heart.

The aim of our study was to determine the main ionic mechanisms responsible for the electrophysiological alterations of ventricular action potentials associated with cardiac dilation in a strain of cardiomyopathic Syrian hamsters which does not develop hypertrophy during the first five months of life. Right and left ventricular action potentials (APs) were recorded in Langendorff perfused isolated hearts from dilated cardiomyopathic (MS 200) and normal hamsters at 60, 120, and 180 days of age. AP characteristics differed in the two ventricles and in different regions (base, apex) of the left ventricle in both strains. When recorded in a given region (apex), the plateau was always of higher amplitude and longer duration, i.e., of larger area, in diseased as compared to normal hearts. The participation of the calcium-independent 4-aminopyridine (4-AP) sensitive transient outward current, Ito1, in the left ventricular AP plateau repolarization was smaller in dilated than in control hearts at any age and AP area was the same in both strains at 60 days of age in the presence of 4-AP. The participation of the cadmium (Cd) sensitive L-type Ca current was investigated in the development of AP plateau at 120 days of age and was smaller in dilated than in control hearts. The participation of the Na-Ca exchange inward current, INa-Ca, in the development of the AP plateau was similar in both strains at 60 days of age; later on, it strongly decreased in control hearts, whereas it remained high in diseased hearts. The tetrodotoxin sensitive slowly inactivating inward current was not increased in dilated hearts compared to control hearts. Our results show that the AP lengthening observed, in dilated non-hypertrophic hamster hearts, results essentially from a reduced participation of Ito1 at 60 days of age, whereas it results from both a reduced participation of Ito1 and an increased participation of INa-Ca at 120 and 180 days of age.

[Transient outward potassium current and repolarization of cardiac cells]. / Courant potassique transitoire sortant et repolarisation des cellules cardiaques.

The transient 4-aminopyridine-sensitive outward potassium current, Ito, is one of the ionic membrane currents involved in the repolarization of cardiac action potentials. It is present in several species (rat, dog, human) but not in guinea pig ventricle. It induces both a marked lowering of the ventricular action potential plateau level and an early repolarization wave in the ventricular ECG complex of hypothermic rats. In dog ventricle where Ito is much shorter than the action potential plateau it can induce only a transient initial repolarization (notch). The distribution of Ito is heterogeneous across the dog left ventricular free wall, the current being of sizeable amplitude in epicardial and midmyocardial layers but absent in the endocardial layer. As a result, ventricular action potentials exhibit a notch only in epicardial and mid layers. Although the physiological role of Ito remains unclear, we suggest that it can participate in the control of calcium current intensity by influencing the level of the initial part of the plateau. In pathophysiological conditions, Ito may exert unfavourable effects, specially during simulated ischemia when the notch reaches the cellular repolarization threshold, thus inducing premature termination of the action potential, an obvious cause of drastic electrical heterogeneity and resulting severe arrhythmias. The current Ito is reduced in moderate cardiac hypertrophy and dilatation and almost entirely suppressed in severe hypertrophy. Ito is of larger amplitude in human atrial than in ventricular myocytes. The heterogeneous distribution of Ito described in the dog has also been found in human ventricles. Because Ito is markedly prolonged at low temperatures it is suggested that it can be responsible for the early repolarization wave (J wave) observed in the ECG of subjects submitted to hypothermia.

[Electrogenic sodium-calcium exchange and electrical activity in cardiac cells]. / Echange sodium-calcium électrogène et activité électrique cellulaire cardiaque.

Several studies have shown that the Na-Ca exchange is electrogenic in cardiac tissues and that the current carried by the exchange, iNa-Ca, participates in the development of the cardiac action potential plateau. The aim of the present study was to assess the contribution of iNa-Ca to the development of the plateau in different preparations, i.e. normal and hypertrophied (DOCA-salt) perfused rat hearts, normal and dilated (MS 200 strain) perfused hamster hearts and normal human atrial myocytes and to examine the repercussion of iNa-Ca suppression on the global electrogram of the perfused guinea-pig heart. iNa-Ca was suppressed by briefly substituting lithium for sodium (Li test). In the normal rat heart, the Li test resulted in a depression of the late component of the plateau. In the severely hypertrophied rat heart, the action potential was lengthened, the two components of the plateau barely distinguishable. The part of the plateau suppressed during Li perfusion was not larger in amplitude than in the normal rat heart, but was of much longer duration. In the dilated heart of hamsters older than 60 days of age the shortening effect of the Li test was much larger than in the normal hamster heart indicating a strongly increased contribution of iNa-Ca to the plateau development during dilatation. The contribution of iNa-Ca to the action potential plateau of human atrial myocytes was as large as that found in animal ventricles and varied with the type of cell studied. In the perfused guinea-pig heart the Li test resulted in a sizeable shortening of QT duration and a marked decrease in T wave amplitude suggesting that iNa-Ca may be a major source of current in the building up of the ventricular complex of the electrocardiogram in normal and pathological conditions.

Ionic basis of the action potential prolongation in ventricular myocytes from Syrian hamsters with dilated cardiomyopathy.

OBJECTIVE: The aim of our study was to determine the main electrophysiological alterations associated with cardiac dilation in MS200 strain Syrian hamsters, a model of genetically determined cardiomyopathy. METHODS: Ventricular action potentials (APs) were recorded with standard microelectrodes in isolated hearts from 120-day-old cardiomyopathic (strain MS200) and age-matched control (strain CHF148) Syrian hamsters. Ionic currents were recorded from single ventricular myocytes using the whole-cell patch-clamp technique. RESULTS: In MS200, AP was prolonged and the plateau phase was markedly increased as compared to CHF148. Differences in both AP duration and 4-aminopyridine-induced AP lengthening between epicardial and endocardial tissues were less marked in MS200 than in CHF148 ventricles. Cell size and membrane capacitance were not higher in MS200 than in CHF148 myocytes, indicating the absence of cell hypertrophy in myopathic ventricles. The L-type calcium current (ICa,L) density was significantly reduced in MS200 and the voltage-dependence of both steady-state activation and inactivation was altered. The voltage-dependent outward current was composed of both transient (Ito1) and sustained (Iss) components, respectively sensitive and insensitive to 4-aminopyridine. Ito1 density was strongly depressed in MS200 compared to CHF148, whereas Iss density was only slightly reduced. The conductance-voltage and steady-state inactivation relationships for Ito1 were shifted to more positive potentials in MS200. The Ito1 recovery process was markedly slower in MS200 than in CHF148. The steady-state current-voltage relationships, in the physiological voltage range, were superimposable in MS200 and CHF148. CONCLUSIONS: In ventricular myocytes from dilated heart of MS200 Syrian hamsters, Ito1 is more drastically depressed than ICa,L. Such an observation might partially explain dilation-induced AP lengthening.

Depressed transient outward current density in ventricular myocytes from cardiomyopathic Syrian hamsters of different ages.

We determined whether the dilated cardiomyopathy which develops between 30 and 140 days of age in the Syrian hamster strain MS200, before the onset of cardiac hypertrophy and failure, is associated with alterations in both the action potential (AP) and the Ca(2+)-independent transient outward current, Ito1. AP was recorded in perfused hearts using microelectrodes and Ito1 was recorded in single ventricular myocytes using the whole-cell patch-clamp. The MS200 strain was compared to the control CHF148 strain at different periods of age (60, 90, 120 and 180 days). APs were markedly lengthened in MS200 compared to CHF148 hearts at all ages studied. Cell membrane capacitance increased with age in the two strains, but was not significantly higher in MS200 than in CHF148 of a given age, indicating the absence of cell hypertrophy. At 60 days of age, Ito1 density was the same in the two strains. Later on, Ito1 density increased markedly at 90-120 days then decreased at 180 days in CHF148, whereas this increase was delayed and of reduced amplitude in MS200. The sustained component of outward current, Isus, was not sizeably different in the two strains. The conductance-voltage and steady-state inactivation relationships were shifted with age towards positive potentials by 15 mV in the two strains, but earlier in MS200 (90 days) than in CHF148 (180 days). Similarly, the recovery of Ito1 from inactivation exhibited a slow component which increased with age in the two strains but was larger in MS200 than in CHF148. In conclusion, alterations of Ito1 may contribute to changes in shape of AP, but cannot entirely explain dilation-induced AP lengthening.

Depressed transient outward and calcium currents in dilated human atria.

OBJECTIVE: The aim was to compare action potentials and ionic currents (steady state current, calcium current, calcium independent transient outward current) in two groups of trabeculae and myocytes, isolated from either dilated or non-dilated human atria. METHODS: Specimens of right atrial appendage were obtained from two groups of adult patients at the time of open heart surgery, a group with non-dilated atria and a group in which right atria were clearly dilated. Action potentials were recorded with standard microelectrodes from isolated superfused trabeculae. Action potentials and ionic currents were recorded from single myocytes using the patch clamp technique in the whole cell configuration in current clamp and voltage clamp modes respectively. RESULTS: In trabeculae taken from dilated atria the action potential was shortened and the plateau was markedly depressed compared to trabeculae taken from non-dilated atria. Similar results were obtained with single myocytes isolated from non-dilated and dilated atria. The density of the steady state current measured at the end of 0.75 s or 1 s pulses was not statistically different in the two groups of cells in the whole range of negative potentials, whereas at strongly positive potentials (> +40 mV) it was significantly reduced in cells from dilated atria compared to cells from non-dilated atria. The density of the total peak outward current was significantly reduced in cells from dilated atria [13.46(SEM 2.7) pA.pF-1 at +70 mV, n = 18] compared to cells from non-dilated atria [33.12(6.2) pA.pF-1, n = 20, p < 0.001]. The transient component of outward current was strongly depressed (at +20 mV and more positive potentials) in cells from dilated atria. The calcium current density was still more severely depressed than the total outward current in cells from dilated atria [4.46(1.06) pA.pF-1 at +20 mV, n = 26] compared to cells from non-dilated atria [17.43(1.98) pA.pF-1, n = 38, p << 0.001]. Kinetic parameters of both calcium and transient outward currents remained similar in cells from the two groups. CONCLUSIONS: The observation that in cells from dilated human atria the calcium current is more severely depressed than the total outward current can help to explain why in dilated human atria the action potential plateau is shorter and of lower amplitude than in non-dilated atria.

Pro-arrhythmic effect of nicorandil in isolated rabbit atria and its suppression by tolbutamide and quinidine.

Nicorandil, a potent vasodilator substance which exerts its effects through complex mechanisms including KATP channel activation, has so far been reported to exert antiarrhythmic but not pro-arrhythmic cardiac activity. We now examined the effects of 10(-4) M nicorandil on spontaneously active or electrically driven isolated rabbit atria. Nicorandil (a) significantly reduced the action potential duration at both 50% (by approximately 45%) and 80% (by approximately 30%) repolarization and the effective refractory period (by approximately 25%) and (b) reproducibly induced short periods of tachycardia either in normal Tyrode solution after a single extra-stimulus or in low-potassium media in the absence of extra-stimulation. Quinidine (10(-5) M) or the KATP channel inhibitor, tolbutamide (10(-5) M), suppressed the nicorandil-induced arrhythmias. It is suggested that the pro-arrhythmic effect of nicorandil results from its KATP channel opener activity and occurs essentially when the underlying conditions facilitate re-entry.

Differential effects of quinidine and flecainide on plateau duration of human atrial action potential.

Quinidine and flecainide, two class-I antiarrhythmics increase action potential duration (APD) at 90% repolarization and cellular refractory period in human atrial fibers without significant change in resting potential. On the other hand, quinidine decreases APD at 50%, whereas flecainide slightly increases, which suggests different effects on Ca2+ current. Using isolation cell procedure and whole cell recording, we found that 10 microM quinidine (34.77 +/- 6.5%, n = 5) and 0.5 microM flecainide (50.46 +/- 6.2%, n = 4) decrease calcium current in human atrium. It is concluded that, at therapeutical concentrations, quinidine and flecainide modify the action potential plateau phase in a different manner, which is not only related to the calcium current decrease.

Effects of atrionatriuretic factor on Ca2+ current and Cai-independent transient outward K+ current in human atrial cells.

The effect of 10 nM atrial natriuretic peptide (ANF) on macroscopic L-type calcium current, ICa, and calcium-independent outward potassium current, Ilo, were studied in myocytes isolated from human atrial trabeculae using the whole-cell-recording patch-clamp technique. When cells were dialysed with pipette media containing 0.2 mM GTP, ANF reduced ICa by 37.81% +/- 5.4% at +20 mV and Ilo by 21.72% +/- 3.68% at +60 mV in a reversible manner. When ICa was increased by beta-adrenoreceptor stimulation (0.1 microM isoproterenol) or by the phosphodiesterase inhibitor isobutylmethylxanthine (10 microM) ANF reduced ICa by 24.99 +/- 3.4% and by 39.9 +/- 6.3% respectively. In cells dialysed with GTP-free pipette media, ANF increased ICa markedly (39.8% +/- 7%) and reversibly, whereas it still depressed Ilo (18.92% +/- 2%). Addition of 0.2 mM GTP[gamma S] to the pipette solution in the absence of GTP increased ICa, decreased Ilo and suppressed the effect of ANF on both ICa and Ilo. It is suggested that activation of the ANF receptor in human atrial cells reduces ICa via guanylate-cyclase-dependent cGMP production, increases ICa via Gs protein activation and decreases Ilo via Gi protein activation.

Calcium current depression in isolated human atrial myocytes after cessation of chronic treatment with calcium antagonists.

The present study investigates the possibility that the slow calcium current of human atrial cardiac cells is modified by chronic treatment (1-24 months) with calcium antagonists (nifedipine, nicardipine, or diltiazem) in a manner different from a simple drug-induced blocking effect. Data from treated patients were recorded approximately 30 hours after cessation of the treatment and were compared with those of nontreated patients. In the treated group, the action potential plateau of atrial fibers was always markedly and irreversibly depressed, and action potential duration measured at 50% repolarization was markedly shortened (81 +/- 12 msec, n = 13) compared with normal values (155 +/- 9 msec, n = 28). In isolated atrial cells, peak calcium current density at +10 mV in treated cells was more than three times as small as that in nontreated cells. Steady-state inactivation relations of calcium current as a function of membrane potential were not significantly different in treated and nontreated cells. In contrast, in treated and nontreated cells superfused with 10(-6) M nifedipine, the curves were markedly shifted toward negative potentials. Cell superfusion with 10(-6) M Bay K 8644 increased calcium current to a larger extent in nontreated cells (sixfold increase) than in treated cells (threefold increase), whereas a 23-fold increase was observed in nontreated cells in which the current had been previously depressed by superfusion with 10(-6) M nifedipine. In contrast to Bay K 8644, 10(-7) M isoproterenol and 10(-8) M angiotensin II increased the calcium current to the same extent in both treated and nontreated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 2,3-butanedione 2-monoxime on slow inward and transient outward currents in rat ventricular myocytes.

The effect of 2,3-butanedione 2-monoxime (BDM), a substance possessing phosphatase-like activity, was studied on action potentials of isolated rat heart and on the slow inward calcium current and outward current (including the 4-aminopyridine (4-AP)-sensitive transient outward component), in rat ventricular myocytes. In contrast to what was observed by other authors in different species and cardiac tissues, BDM increased markedly the amplitude and duration of the rat ventricular action potential plateau. On the other hand, in the presence of 4-AP and ryanodine BDM shortened action potential duration. BDM decreased in a dose dependent manner the amplitude of both the slow inward calcium current and the transient outward current, accelerated their inactivation and shifted their steady-state inactivation-voltage relationships towards negative potentials. BDM also depressed other components of outward current. It is suggested that the lengthening effect of BDM on action potential duration results mainly from the simultaneous reduction of both the slow inward calcium current and the transient outward current, two antagonistic currents with unequal influences on action potential plateau development. The similarity of effect of BDM on these two currents also suggests that ionic channels generating them might require similar phosphorylation for their functioning.

Stimulatory effect of ouabain on T- and L-type calcium currents in guinea pig cardiac myocytes.

The effect of 10(-6) M ouabain on macroscopic low-threshold T-type Ca2+ and high-threshold L-type Ca2+ currents was studied by whole cell recording in isolated guinea pig ventricular myocytes superfused with K-free, Na-free media, i.e., after suppression of Na-K-ATPase activity and Na influx through the Na-Ca exchanger. Under such conditions, the amplitudes of the two currents were significantly increased by ouabain. In particular, the current occurring in the -50 to -20 mV range (T-type Ca2+) was increased two- to threefold by ouabain and suppressed by 40 microM Ni2+. Ouabain shifted by approximately 10 mV toward negative potentials the steady-state inactivation curve of the T-type Ca2+ current but not that of the L-type Ca2+ current. It is concluded that ouabain enhances not only L-type Ca2+ current but also T-type Ca2+ current possibly through different mechanisms.

Effect of maitotoxin on calcium current and background inward current in isolated ventricular rat myocytes.

Maitotoxin (MTX) irreversibly suppressed the voltage-dependent calcium current after a variable delay, an effect which was preceded, in 61% of the cells, by a transient increase in calcium current partly attributable to a shift (4-7 mV) of the activation curve towards negative potentials. MTX also induced the development of a voltage-independent background inward current which did not occur in the absence of external calcium and was reduced by removal of external sodium, by calcium channel blockers and by high concentrations of quinidine. MTX-induced single channel activity consisted of long lasting bursts of inward current. Channel activity was voltage-independent, with a unitary conductance of 14 pS and an extrapolated reversal potential of +16 mV. Single-channel current amplitude was not detectably reduced in the absence of external calcium but strongly reduced in the absence of external sodium, in the presence of 2 mM nickel or when external sodium was replaced by 96 mM calcium or 50 mM barium. The channel activity was also inhibited by quinidine. It is concluded that MTX alters, then suppresses the voltage-activated calcium current and induces the development of a voltage-independent inward current, part of which results from the opening of nickel-sensitive cation channels, mostly permeable to sodium ions.

Effects of activation of ATP-sensitive K+ channels in mammalian ventricular myocytes.

A cromakalim analogue, SR 44866, is shown to open ATP-sensitive K+ channels in ventricular myocytes. The channels opened by SR 44866 were closed by internal ATP and had the same current-voltage relationship as ATP-sensitive K+ channels; channels closed by ATP could be opened by SR 44866. SR 44866 was effective when applied to either side of excised membrane patches and when included in the pipette during cell-attached membrane recordings. SR 44866 also opened channels in cell-attached membrane patches when it was applied to the cell membrane outside of the pipette. The current evoked by SR 44866 in whole cell recordings was inhibited by tolbutamide. SR 44866 reduced the duration of action potentials and the amplitude and duration of evoked muscle contractions in guinea pig papillary muscle, with dissociation constants of 6.8 microM at 24 degrees C and 1.9 microM at 34 degrees C and Hill coefficients of 1.72 and 1.71, respectively. We conclude that the opening of ATP-sensitive K+ channels has profound inhibitory effects on the electrical and mechanical activity of cardiac muscle.

Comparative effects of three class I antiarrhythmic drugs on plateau and pacemaker currents of sheep cardiac Purkinje fibres.

The electrophysiological effects of three class I antiarrhythmic drugs, lignocaine, disopyramide, and penticainide, were compared in sheep cardiac Purkinje fibres. Action potential duration was shortened with all three drugs, the effect being small with disopyramide, moderate with penticainide, and greatest with lignocaine. Slowing down of the early phase of repolarisation was greatest with disopyramide, smaller with penticainide, and did not occur with lignocaine. Automaticity recorded in low potassium media was unaltered (disopyramide), depressed (penticainide), or stopped (lignocaine). Ionic currents were recorded in short fibres using the two microelectrode voltage clamp technique. The tetrodotoxin sensitive slow component of the sodium current was suppressed (lignocaine) or reduced (penticainide and disopyramide) and the instantaneous background potassium current slightly reduced (disopyramide more than penticainide), unaffected, or slightly increased (lignocaine). The three drugs depressed moderately and similarly the slow inward calcium current. The amplitude of the 4-aminopyridine sensitive transient outward potassium current was almost unaffected (lignocaine) or appreciably depressed (disopyramide more than penticainide). The pacemaker current was reduced greatly by lignocaine, moderately by penticainide, and slightly by disopyramide. Changes in ionic currents may explain the effects of the three drugs on action potential plateau and automaticity of sheep Purkinje fibres. It is concluded that the pronounced differences observed in the effects of these three class I antiarrhythmic drugs on pacemaker depolarisation and on initial repolarisation may justify a more discriminative subdivision of class I antiarrhythmic drugs.

Effect of 3-5-3'triiodothyronine treatment on cardiac action potential of streptozotocin-induced diabetic rat.

Electrical activity of rat atrium of streptozotocin-diabetic and control rats was compared. (i) As occurs in the ventricle, diabetes lengthens the cardiac atrial action potential. (ii) Treatment by T3 of diabetic animals decreases action potential duration to normal values and causes partial recovery in plateau decay during the late phase of repolarization. (iii) T3 treatment however, does not completely normalized the action potential of the diabetic rat atrium, which remains abnormal during the early phase of repolarization. These results demonstrate that some defects in membrane mechanisms involved in the early phase of action potential repolarization are attributable solely to diabetes. The possible nature of these mechanisms is discussed.

Two types of transient outward currents in adult human atrial cells.

It has been suggested in a previous article [Escande et al., Am. J. Physiol. 249 (Heart Circ. Physiol. 18): H843-H850, 1985] that transient outward currents may participate in the initial repolarization of human atrial fibers. The present study substantiates the existence of such currents in human myocardium. Membrane currents were recorded in enzymatically dissociated cells using the whole cell patch-clamp technique. Two kinds of transient outward currents were observed: 1) a long-lasting outward current, (ilo), which was suppressed by 4-aminopyridine. The time to peak of ilo was 18.0 +/- 0.7 ms, and its inactivation time constant was 35.7 +/- 2.1 ms at room temperature (test pulses, +20 mV; holding potential, -40 mV); 2) a brief outward current (ibo), which persisted with 3 mM 4-aminopyridine and exhibited a shorter time to peak (5.5 +/- 0.2 ms) and a faster decay (time constant, 9.1 +/- 1.8 ms). ilo was inhibited by Ba but was insensitive to the calcium blocker Co. Co blocked both the slow inward current (isi) and ibo. It is concluded that two different transient outward currents control the repolarization in human atrial cells.