Taurine modulates I_Kr but not I_Ks in guinea-pig ventricular cardiomyocytes

Abstract

1. Effects of taurine on the delayed rectifier K⁺ current (I_K) in isolated guinea-pig ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentration ([Ca²⁺]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at 36°C.

2. Addition of taurine (10–20 mM) decreased the action potential duration (APD) at pCa 8, but increased the APD at pCa 6. Taurine (20 mM) enhanced I_K at 70 mV by 22.4±3.1% (n=6, P<0.01) at pCa 8, whereas taurine inhibited the I_K by 27.1±2.7% (n=6, P<0.01) at pCa 6. These responses behaved in a concentration-dependent manner.

3. The I_K is composed of the rapid and slow components (I_Kr and I_Ks). When [Ca²⁺]_i was pCa 6, taurine at 20 mM reduced the tail current of I_Kr at 70 mV by 16.5±2.7% (n=5, P<0.05) and that of I_Ks at 70 mV by 27.1±2.8% (n=6, P<0.01). In contrast, at pCa 8, the tail currents of I_Kr and I_Ks at 70 mV were enhanced by 13.4±3.2% (n=7, P<0.05) and by 22.4±3.1% (n=7, P<0.01), respectively. The voltages of half-maximum activation (V_1/2) for I_Kr and I_Ks were not modified by taurine.

4. Addition of E-4031 (5 μM) to taurine had a complete blockade of the tail current of I_Kr, but not I_Ks. The remained tail current (I_Ks) in the presence of E-4031 (5 μM) was not affected by taurine (20 mM), but was blocked by 293B (30 μM).

5. These results indicate that taurine modulates I_Kr but not I_Ks, depending on [Ca²⁺]_i, resulting in regulation of the APD.

Introduction

The outward K⁺ currents play an important role in the regulation of repolarization of the action potential in cardiac cells. The blockade of K⁺ channels prolongs the action potential duration (APD) but the stimulation shortens APD. Of the K⁺ currents, the delayed rectifier K⁺ current (I_K) mainly affects APD, regulated strongly by the intracellular or extracellular Ca²⁺ concentrations ([Ca²⁺]_i or [Ca²⁺]_o) (Meech, 1974; Isenberg, 1975) Satoh (1995) has recently reported that, in embryonic chick cardiomyocytes, taurine inhibited I_K at high [Ca²⁺]_i, whereas taurine enhanced I_K at low [Ca²⁺]_i.

Taurine (2-aminoethanesulphonic acid) is a sulphur-containing amino acid, and is abundant in cardiac myocardium and other tissues. During ischaemia and cardiac failure, taurine content in the heart decreases, with the depletion correlated with the degree of mechanical dysfunction (Lombardini, 1980; Kramer et al., 1981). This suggests that taurine may be essential to maintain cardiac function. Thus, the actions of taurine on cardiomyocytes have been investigated (Franconi et al., 1982; Failli et al., 1992). Taurine inhibited the TTX-sensitive Na⁺ current (I_Na) (Satoh, 1998a; Satoh & Sperelakis, 1992). Taurine also inhibited the L-type Ca²⁺ current (I_Ca) at pCa 7, whereas taurine enhances I_Ca at pCa 8 (or 10) (Satoh & Horie, 1998; Satoh & Sperelakis, 1993). From these effective actions, taurine appears to play an important role for regulating [Ca²⁺]_i and [Ca²⁺]_o. If so, taurine may possess and exhibit cardioprotective activity related to its ability to alter Ca²⁺ movement (Huxtable & Sebring, 1983; Satoh & Sperelakis, 1998).

The aim of the present experiments was to examine the modulations of I_K by taurine, using adult cardiomyocytes at different [Ca2+]_i levels (pCa 6 and 8). Recently, the I_K has shown to be composed of rapidly and slowly activated currents (I_Kr and I_Ks) (Sanguinetti & Jurkiewicz, 1990). So, the effects of taurine on I_Kr and I_Ks and the modulation of the APD were also examined.

Methods

Cell preparation

Cells were prepared from tissue taken from the ventricle muscle of guinea-pig hearts, using methods similar to those described previously (Satoh, 1998b; Satoh & Sperelakis, 1992, 1993). Under anaesthesia with sodium pentobarbital (30 mg kg⁻¹, i.p.), the chest was opened and the aorta was cannulated in situ. The heart was dissected out and perfused with normal Tyrode solution on the Langendorff apparatus. After a washout of blood, the heart was perfused with Ca²⁺-free Tyrode solution, and spontaneous beating ceased. Then, the perfusate was switched to low-Ca²⁺ (30–60 μM) Tyrode solution containing 0.4 mg ml⁻¹ collagenase (Type I, Sigma Chemical, St. Louis, MO, U.S.A.) for about 20 min. The heart was washed out by high-K⁺ and low-Cl⁻ solution (KB solution), and was dissected with scissors. The temperature of all solutions was maintained at 36°C.

Whole-cell voltage-clamp experiments

Whole-cell voltage-clamp recordings were performed using an Axopatch patch-clamp amplifier (Axon Instruments, Burlingame, CA, U.S.A.) and standard techniques. Patch pipettes from borosilicate glass capillaries were fabricated using a two-stage puller, and had a resistance of 5–7 MΩ for whole-cell experiments. The series resistance error was less than 3–7 mV, and no compensation was used. The liquid junction potential between the pipette solution and the external solution (less than 10 mV) was corrected for all membrane potential recording. Experiments were carried out at temperature of 36°C. The data were stored and analysed on an IBM-AT microcomputer, using the PCLAMP analysis program (Axon Instruments). Current traces were filtered using a cut-off frequency of 1 KHz for plotting. All values are given as means±s.e.mean. The differences of the mean values were analysed by Student's t-test and analysis of variance (ANOVA) for paired data, and a P value less than 0.05 was considered significant.

Experimental solutions

The composition of the modified Tyrode solution was (in mM): NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, NaH₂PO₄ 0.3, glucose 5.0, and HEPES 5.0. The pH was adjusted to 7.4 with NaOH. To avoid the interference of other currents, 1 μM nicardipine was added to the external Tyrode solution to block the L-type Ca²⁺ current. Taurine (Sigma Chemical Co., St. Louis, MO, U.S.A.) was dissolved to the desired concentrations directly in the bath solution, and the solution was superfused. Also, E-4301 (Eizai Pharmaceutical Co., Tokyo, Japan), a blocker of I_Kr, and 293B (Hoechst Pharmaceutical Co., Germany), a blocker of I_Ks, were used. The pipette solution (intracellular) contained (in mM): K-aspartate 110, KCl 20, MgCl₂, EGTA 10, Mg-ATP 5, creatine phosphate 5, and HEPES 5 (pH 7.2). The concentrations of free Ca²⁺ (pCa 8 and 6) in the internal solution were calculated on the basis of apparent stability constants for EGTA, Ca²⁺-ATP, Ca²⁺-creatine phosphate, Mg²⁺-EGTA, Mg²⁺-ATP, and Mg²⁺-creatine phosphate, according to the calculation of Fabiato & Fabiato (1979) and the correction of Tsien & Rink (1980).

Results

Effects on the action potential

To examine the effect of taurine on the action potential configuration, current-clamp experiments were carried out. The single cell was stimulated at 1 Hz. As shown in Figure 1A,B, taurine (20 mM) markedly shortened the action potential duration (APD) at pCa 8, but prolonged it at pCa 6. The 75% repolarization of APD (APD₇₅) at pCa 8 was shortened by 7.5±1.8% (n=6, P>0.05) and 19.7±2.3% (n=6, P<0.05) in the presence of 10 and 20 mM taurine, respectively (Figure 1C). At pCa 6, taurine at 10 and 20 mM prolonged APD₇₅ by 64.5±2.8% (n=6, P<0.001) and 93.2±4.3% (n=6, P<0.001), respectively. The resting potential was unaffected.

Figure 1.

Effects of taurine on the action potentials in ventricular cardiomyocytes. (A) Action potentials at pCa 8 before and after taurine application. (B) Action potentials at pCa 6. Taurine (10 and 20 mM) was cumulatively added to the bath solution. (C) Time-dependent changes in APD75 at pCa 8 and pCa 6.

Taurine's action on I_K

The I_K current was measured by applying test pulses (2 s duration) to −30 –+70 mV from a holding potential of −40 mV. The I_K current at 90 mV was 16.7±0.5 pA pF⁻¹ at pCa 8 (Figure 2A). The average capacitance was 88.9±3.1 pF (n=27). Cumulative administrations of taurine (10 and 20 mM) to the bath solution enhanced I_K at 70 mV by 13.4±3.2% (n=6, P<0.05) and by 22.4±3.1% (n=6, P<0.01), respectively. Current-voltage (I–V) relations in the absence and presence of taurine (10 and 20 mM) are given in Figure 2B.

Figure 2.

Enhancement of the delayed rectifier K⁺ current (I_K) induced by taurine at pCa 8. (A) Current traces in the absence and presence of 10 and 20 mM taurine. A test pulse (for 2 s) was applied between 0–90 mV from a holding potential of −40 mV. The short line at the left of the current records represents the zero current level. (B) Current-voltage relationship for I_K (n=6).

On the other hand, the I_K at pCa 6 was inhibited by application of taurine in a concentration-dependent manner (Figure 3A,B). The I_K and its tail current were strongly activated as compared with those at pCa 8. Figure 3B shows the I–V relationship for I_K. The inhibition at 70 mV in the presence of 10 and 20 mM of taurine was 16.5±2.8% (n=6, P<0.05) and 27.1±2.8% (n=6, P<0.01), respectively. The responses to taurine were incompletely recovered by a 20-min washout. Low concentrations (1–5 mM) of taurine had little or no effect on I_K.

Figure 3.

Inhibition of I_K induced by taurine at pCa 6. (A) Current traces in the absence and presence of taurine. Test pulses for 2 s were applied between −10 –+70 mV from a holding potential of −40 mV. The short line at the left of the current records represents the zero current level. (B) Current-voltage relationship for I_K in control and in 10 and 20 mM taurine (n=6).

Effects on activation curves for I_Kr and I_Ks

To examine the degree of activation of the I_K current, the amplitude of the outward tail current of I_K was plotted along the voltage axis. At pCa 8, 10 and 20 mM taurine enhanced the tail current at 70 mV by 36.2±2.1% (n=5, P<0.01) and 72.3±3.8% (n=5, P<0.01), respectively (Figure 4A). The curves in Figure 4C were fitted by an empirical equation; p=1/{1+exp[(Vm−V_1/2)/S]}. V_1/2 is the potential of half-activation, and S is the slope factor. The slope factor at pCa 8 was 9.8±2.1 (n=5) in the control and 9.9±1.3 (n=5) in the taurine (10 and 20 mM)-treated cells. The V_1/2 value in five cells was 42.3±2.6 mV (n=5) in control and 44.8±2.2 mV (n=5) at 20 mM taurine. The difference was not significant (P<0.05).

Figure 4.

Effects of taurine on the activation process for I_K. Holding potential was −40 mV. (A,B) Outward tail currents in control and in 10–20 mM taurine at pCa 8 (n=5) and 6 (n=6). (C,D) Normalized activation curves for I_K at pCa 8 and 6, respectively. The curve was fitted by Voltzman equation.

At pCa 6, the tail current at 70 mV was inhibited by 12.4±2.0% (n=6, P<0.05) at 10 mM taurine and by 35.1±2.4% (n=6, P<0.01) at 20 mM taurine (Figure 4B). Increasing [Ca²⁺]_i (from pCa 8 to 6) shifted the normalized curve to a more negative potential in normal Tyrode solution; by approximately 10 mV at V_1/2 from the curves in Figure 4C,D. The slope factors were 10.2±0.3 (n=5) in the control, and 10.8±0.4 in the presence of taurine (10 and 20 mM). The V_1/2 value was 32.3±2.2 mV (n=5) in the control, and 30.1±2.8 mV (n=5) in the presence of 20 mM taurine; the difference was not significant (P<0.05).

The I_K activated by the long test pulse is composed of rapidly and slowly activated currents (I_Kr and I_Ks). Effects of taurine on the tail current of I_Kr were examined by test pulse for 200 ms duration to separate the I_Ks component. Taurine had the stimulatory effects on the tail current of I_Kr at 50 mV at pCa 8 and the inhibitory effects at pCa 6 (Figure 5A,B). The tail currents are plotted in Figure 5C,D. Additional application of E-4031 (5 μM) blocked I_Kr completely. The V_1/2 values were −10.2±0.8 mV (n=5) at pCa 8, and 4.3±0.9 mV (n=5) at pCa 6. Taurine did not affect the V_1/2 values at both pCa levels (Figure 5E,F).

Figure 5.

Modulation by taurine of the tail current and the activation curve for rapid component (I_Kr) of I_K. (A,B) Outward tail currents for 200 ms in control and 20 mM taurine at pCa 8 and 6. At pCa 6, the current traces during test pulses disappeared through saturation. (C,D) Peak of the tail currents (n=5) at pCa 8 and 6. (E,F) Normalized curves of the activation for I_Kr at pCa 8 and 6 respectively.

Effects of taurine on the tail currents of I_Kr activated by less 200 ms duration and on that of I_K (or I_Ks) by 1 or 2 s duration were examined. Taurine (20 mM) inhibited the tail current of I_Kr, as shown in Figure 6A. The inhibition was 35.7±2.8% (n=6, P<0.05) at 10 mM and 56.0±2.3% (n=6, P<0.01) at 20 mM taurine. Addition of E-4031 (5 μM) blocked the I_Kr completely, but not I_K fully. The effects on I_Kr at pCa 8 and 6 are summarized in Figure 6B. The tail current was blocked by E-4031 (5 μM). Taurine had little or no effect on the remained tail current, that is I_Ks, in the presence of E-4031 (5 μM) at both pCa levels. However, addition of 293B (30 μM) had almost blockade of I_Ks (Figure 6C).

Figure 6.

Effects of taurine on the tail currents of rapid and slow components of I_K (I_Kr and I_Ks). (A) Current traces of I_Kr and I_Ks at pCa 6. Test pulses were applied to 50 mV from −40 mV for 200 ms and 1 s. (B) Percentage changes in peak of I_Kr (n=6) at pCa 8 and 6 by taurine (10 and 20 mM) and E-4031 (5 μM). Ordinate axis is represented control value as 100%. (C) Changes in I_Ks at pCa 8 and 6 by taurine and 293B (30 μM) in the presence of 5 μM E-4031 (n=6).

Discussion

Taurine, an amino acid largely obtained from the diet, has been found to exert many important physiological functions. Its electrical and mechanical actions are independent of many known regulators, such as cyclic AMP and cyclic GMP levels, (Na,K)-ATPase activity and calmodulin-dependent protein kinase action, although taurine may inhibit protein kinase C activity (Segawa et al., 1985; Lombardini, 1992). The experiments were designed to examine the effects of taurine on I_K (I_Kr and I_Ks) at low and high [Ca²⁺]_i in single isolated (adult) guinea-pig ventricular cardiomyocytes. The present experiments showed the following. (1) The APD₇₅ in the presence of taurine decreased at pCa 8, whereas taurine increased it at pCa 6. (2) Taurine enhances I_K at pCa 8 and inhibited I_K at pCa 6. (3) Taurine stimulated the tail current of I_K at low pCa, but depressed it at high pCa. (4) The tail current of I_K was not affected by taurine. (5) The voltage of half-maximum activation of I_Ks was unaffected by taurine at both pCa 8 and 6. (6) The taurine-induced effects were irreversible even after a 30-min washout, consistent with our previous reports (Satoh & Sperelakis, 1992, 1993, 1998). The basis for this is unclear, but some possibilities exist: (a) the recovery from taurine may occur slowly and be not observed by 30 min, and (b) the recovery may be masked by time-dependent damage to the myocytes.

Action potential duration

At the plateau, the inward and outward currents are practically in balance, and a small change in one of the currents will greatly affect the course of the potential. From a theoretical point of view, APD is strongly regulated by the delayed rectifier (and time-dependent activated) K⁺ current (I_K). Taurine stimulated I_K at pCa 8, whereas it depressed I_K at pCa 6. Since the I_K in cardiac cells is dependent on [Ca²⁺]_i and [Ca²⁺]_o (Meech, 1974; Isenberg, 1975), the Ca²⁺-activated I_K might be stimulated through the enhancement of I_Ca even when [Ca²⁺]_i is low. Since, in the present experiments, [Ca²⁺]_i was buffered at pCa 6 or 8 (EGTA 10 mM in the pipette), the actions on I_K induced by taurine would be independent of [Ca²⁺]_i.

The APD may also be modulated by such ionic currents as I_Ca and the fast Na⁺ current (I_Na) (Carmeliet & Saikawa, 1982). Taurine increased I_Ca at low pCa, but decreased I_Ca at high pCa. The inactivation of I_Ca is composed of both fast ([Ca²⁺]_i-dependent) and slow (voltage-dependent) exponentials (Kass & Sanguinetti, 1984; Satin & Cook, 1989). In embryonic chick ventricular myocytes, taurine affected only the slow time constant for I_Ca inactivation, but not the fast time constant (Satoh & Sperelakis, 1993). The increase in the time constant for inactivation would result in APD prolongation. In this study, thus, the Ca²⁺ channel was blocked by Ca²⁺ antagonist. On the other hand, taurine inhibits I_Na, independent of [Ca²⁺]_i level (Satoh, 1998a). The time constant for I_Na inactivation (with a single component) is not modified by taurine (Satoh & Sperelakis, 1992). In this study, the I_Na channel is not activated at −40 mV of holding potential. Therefore, these results suggest that taurine-induced APD modulation is mainly due to its actions on I_K. In addition, taurine inhibits the inwardly rectifying K⁺ current (I_Kl), which may also partly modulate the APD (Satoh, 1998b).

I_K at different pCa levels

In the present experiments, the different pCa levels were used to elucidate the effects of taurine on I_K current. The amplitude of I_K was greater at pCa 6 than at pCa 8, and its activation curve was shifted at V_1/2 by approximately 10 mV to the negative potential (see ">Figures 3B and 4B). The potentiation of I_K amplitude is consistent with previous reports (Meech, 1974; Isenberg, 1975).

Taurine inhibited I_K at pCa 6, but enhanced I_K at pCa 8, consistent with the previous report (Satoh, 1995). Recently, I_K is separated by two distinct components of rapidly and slowly activation (I_Kr and I_Ks) (Sanguinetti & Jurkiewicz, 1990). The I_Kr is selectively E-4031-sensitive and is activated fully within 200 ms. Taurine markedly affected I_Kr. In the presence of E-4031, however, taurine had little or no effect on the remained current (I_Ks), and addition of 293B (a selective I_Ks inhibitor) (Suessbrich et al., 1996) blocked it completely. Therefore, these results indicate that the dual action of taurine at different pCa levels is mostly due to its effects on I_Kr, but not I_Ks. The selective inhibition would somewhat play an important role for cardioprotective actions of taurine.

Cardioprotection

Our recent experiments showed that taurine possesses a potent cardioprotective action in embryonic chick cardiomyocytes and guinea-pig ventricular myocytes (Satoh & Sperelakis, 1992, 1998; Satoh, 1998a,1998b). Taurine enhanced I_Ca at low pCa, but inhibited I_Ca at high pCa. Sperelakis and colleagues (Sperelakis & Satok, 1993; Sperelakis et al., 1992, 1988) concluded that one of taurine's actions is to normalize Ca²⁺ movement through the sarcolemma, dampening the effects of variation in perfusate Ca²⁺ concentration. These results indicate that taurine acts on I_Ca in a manner to keep [Ca²⁺]_i levels relatively constant, and consequently plays an important role in maintaining cell viability and cardiac function.

The decline in [Ca²⁺]_i induced by taurine should lead to a reduction of cellular calcium overload during ischaemia. Taurine also promotes the sudden emergence of a dormant I_CaT, contributing to the generation of pacemaker activity during diastole (Satoh & Sperelakis, 1993). Since automaticity is depressed under calcium overload (Satoh & Hashimoto, 1988; Satoh et al., 1989), the stimulation of I_CaT may also be one of the protective actions of taurine. Furthermore, taurine stimulates the hyperpolarization-activated inward current (that is a pacemaker current, I_f) (unpublished observation). In the present experiments, high pCa itself shortened APD, whereas low pCa itself prolonged APD. Extreme prolongation or shortening of APD may alter the refractory period and elicit arrhythmias (Satoh, 1993b). The antiarrhythmic actions of taurine are also produced by the inhibitions of such as the fast Na⁺, Ca²⁺, and the ATP-sensitive channels. These findings demonstrate that the effects induced by high and low [Ca²⁺]_i are antagonized and many ionic channels were modulated by taurine application, indicative of a cardioprotective action of taurine. Although many of taurine's actions on heart still remain unclear, further studies should reveal possible therapeutic uses of taurine.

Abbreviations

ANOVA

analysis of variance

APD

action potential duration

APD₇₅

75%repolarization of action potential duration

[Ca²⁺]_i

intracellular Ca²⁺ concentration

[Ca²⁺]_o

extracellular Ca²⁺ concentration

I_K

delayed rectifier K⁺ current

I_K1

inwardly rectifying K⁺ current

I_Kr

rapid component of delayed rectifier K⁺ current

I_Ks

slow component of delayed rectifier K⁺ current

I_Ca

L-type Ca²⁺ current

I_Na

fast Na⁺ current

S

slope factor

TTX

tetrodotoxin

V_1/2

voltage of half-maximum activation

V_m

membrane potential