Acute exposure to thyroid hormone increases Na⁺ current and intracellular Ca²⁺ in cat atrial myocytes

Abstract

Whole-cell recording methods and fluorescence microscopy were used to study the effects of acute exposure to thyroid hormone (T₃) on cat atrial myocytes. Acute exposure (≈5 min) to 10 nm T₃ significantly increased tetrodotoxin (TTX)-sensitive inward Na⁺ current (I_Na) at voltages between −40 and +20 mV. At maximal I_Na activation (−40 mV) T₃ increased peak I_Na by 32 %. T₃ had no effect on the time course of I_Na decay, voltage dependence of activation, inactivation, or recovery from inactivation. Comparable exposures to reverse T₃ (rT₃) or T₄ had no effect on I_Na. L-type Ca²⁺ current was unaffected by acute exposure to T₃. T₃-induced increases in I_Na were unaffected by 50 μm nickel, a blocker of T-type Ca²⁺ current. T₃ significantly increased cell shortening (+62 %) and could elicit spontaneous action potentials arising from Ca²⁺-mediated after-depolarizations. T₃ (but not rT₃) significantly increased baseline intracellular Ca²⁺, release of Ca²⁺ from sarcoplasmic reticulum (SR) and caffeine (10 mm)-induced release of SR Ca²⁺. We conclude that acute T₃ exposure increases Na⁺ influx via I_Na and thereby stimulates reverse-mode Na⁺-Ca²⁺ exchange to increase intracellular Ca²⁺ content and release. As a result, T₃ increases contraction strength, and can initiate Ca²⁺-mediated arrhythmic activity. Acute non-genomic effects of T₃ can contribute to the positive inotropy and sinus (atrial) tachycardia traditionally attributed to chronic, genomic effects of elevated thyroid hormone on atrial muscle.

Clinically, patients with hyperthyroidism experience elevated heart rate and cardiac contractility with increased stroke volume and cardiac output (Klein & Levey, 2000; Klein & Ojamaa, 2001). Sinus tachycardia at rest and during exercise is the most consistent rhythm disturbance in patients with thyrotoxicosis, with 5-10 % of patients exhibiting atrial fibrillation (Klein & Levey, 2000). The chronic effects of triiodothyronine (T₃), the biologically active form of thyroid hormone, are mediated via nuclear receptors that allow the hormone-dependent regulation of transcription (Brent, 1994). The phenotypic changes that result from T₃ action on the heart enhance cardiomyocyte contractile function and ion channel properties (Freedberg et al. 1970; Klein & Ojamaa, 2001). In addition, hyperthyroidism enhances sarcoplasmic reticulum (SR) Ca²⁺ uptake and release by increasing protein content of SR Ca²⁺-activated ATPase (SERCA2) and the ryanodine receptor while decreasing phospholamban (Kiss et al. 1995; Shenoy et al. 2001). Chronic T₃ exposure also increases slow inward Ca²⁺ current (Binah et al. 1987; Rubinstein & Binah, 1989), outward rectifying K⁺ current (I_K) (Rubinstein & Binah, 1989) and transient outward K⁺ current (Shimoni & Severson, 1996; Sun et al. 2000).

Acute exposure to T₃ also is cardioactive via non-genomic mechanisms of action (Davis & Davis, 1993). T₃ receptors are present in cardiac plasma membranes (Segal, 1990). Acute T₃ exposure slows inactivation of fast Na⁺ current in neonatal cardiac myocytes (Harris et al. 1991), increases single channel Na⁺ current in rat ventricular myocytes (Dudley & Baumgarten, 1993), and increases cellular Ca²⁺ uptake in rat heart slices (Segal, 1990) and isolated perfused heart (Gotzsche, 1994). Although it is well known that hyperthyroid states exert profound effects on atrial function, the vast majority of research has been performed on ventricular muscle. Consequently, less is known about the acute effects of T₃ on atrial myocytes. The purpose of the present study, therefore, was to determine whether acute exposure to T₃ exerts non-genomic effects on atrial myocytes that may contribute to the effects of thyroid hormone traditionally attributed to chronic, genomic mechanisms. The present results indicate that acute exposure of atrial myocytes to T₃ increases I_Na, resulting in elevation of intracellular Ca²⁺ mediated by Na⁺-Ca²⁺ exchange. As a result, T₃ exerts positive inotropic effects and can contribute to the development of Ca²⁺-mediated atrial tachy-dysrhythmias.

Methods

Methods used to isolate atrial myocytes from cat heart have been published in detail previously (Wang & Lipsius, 1995). Adult cats of either sex were anaesthetized with sodium pentobarbital (50 mg kg⁻¹, intraperitoneal). Following a bilateral thoracotomy, the heart was rapidly excised and attached to a Langendorff perfusion apparatus. Hearts were perfused with enzyme (0.06 % collagenase; Worthington Biochemical, type II) to disperse individual cells. Cell isolation was performed on the morning of each experiment.

Cells used for study were transferred to a small (0.4 ml) tissue bath on the stage of an inverted microscope. Cells selected for study were elongated and quiescent, and superfused at ≈5 ml min⁻¹ with a modified Tyrode solution containing (mm): NaCl 137, KCl 5.4, MgCl₂ 1, CaCl₂ 2, Hepes 5, glucose 11, titrated with NaOH (pH of 7.4). Inward Na⁺ current (I_Na) was recorded in a whole-cell ruptured patch recording configuration at room temperature. The internal pipette solution contained (mm): caesium glutamate 100, CsCl 40, MgCl₂ 1, Na₂-ATP 4, EGTA 10, Hepes 10, titrated with CsOH to a pH of 7.2. In the ruptured patch method, the junction potential (10 mV) measured between the internal pipette and bath solutions was subtracted from all voltage measurements. To better control I_Na, external Na⁺ concentration was reduced by replacement with equimolar TEA-Cl. The external recording solution contained (mm): NaCl 50, TEA-Cl 67, KCl 5.4, MgCl₂ 1, CaCl₂ 2, 20 mm CsCl, Hepes 5, glucose 11, titrated with CsOH to pH 7.35. In addition, I_Na was isolated by adding 5 m verapamil to the external solutions, to block L-type Ca²⁺ current (I_Ca,L), and 2 mm 4-aminopyridine, to block transient outward currents. Even with these modifications, it was not possible to completely control I_Na. Nevertheless, membrane voltage in response to the command pulse did not change between control and test (T₃) and therefore the effects of T₃ on peak I_Na cannot be attributed to changes in clamp voltage. I_Ca,L, action potentials and cell shortening were recorded by a perforated patch (nystatin) method (Horn & Marty, 1988). Nystatin was dissolved in DMSO at a concentration of 50 mg ml⁻¹, and then added to the internal pipette solution to yield a final nystatin concentration of 150 μg ml⁻¹. When recording I_Ca,L in the perforated patch configuration the internal pipette solution contained (mm): caesium glutamate 100, CsCl 40, MgCl₂ 1, Na₂-ATP 4, EGTA 0.5, Hepes 10, titrated with CsOH to a pH of 7.2. When recording action potentials in the perforated patch configuration the internal pipette solution contained (mm): potassium glutamate 100, MgCl₂ 1, Na₂-ATP 4, KCl 40, EGTA 0.5, Hepes 10, titrated with KOH to pH 7.2. Peak I_Ca,L amplitude was measured with respect to steady-state current and was not compensated for leak currents. Action potentials were elicited by 3 ms pulses and unloaded cell shortening was measured using a video-based edge detector (Crescent Electronics). A single suction pipette was used to record action potential (bridge mode) and ionic currents (discontinuous voltage clamp mode) using an Axoclamp 2A amplifier (Axon Instruments, Inc., Foster City, CA, USA). The discontinuous (switch clamp) mode precludes the need to compensate for series resistance. In the voltage clamp mode the amplifier sampling rate was approximately 10-12 kHz. A second scope was used to monitor the duty cycle to ensure complete settling of the voltage transient between samples. Computer software (pCLAMP 8; Axon Instruments, Inc.) was used to deliver voltage protocols, acquire and analyse data.

In general, I_Na was activated by depolarizing voltage clamp steps (80 ms) from a holding potential of −80 mV in 10 mV increments. Peak I_Na was measured with respect to steady-state current and was not compensated for leak currents. The concentration of 10 nm T₃ used in the present study is consistent with serum total T₃ levels found in hyperthyroid patients (Chopra & Sabatino, 2000). Cells were exposed to T₃ until its effects on I_Na stabilized, which usually occurred by approximately 5 min. After establishing the ruptured patch configuration, I_Na amplitude exhibited a ‘run down’ of about 10 % (44 ± 5 to 40 ± 4 pA pF⁻¹; n = 10) that reached a relatively stable level within approximately 5 min. Experiments were begun after this time. Current density (pA pF⁻¹) was obtained by normalizing total current-to-cell capacitance.

Voltage dependence of I_Na activation was determined by measuring tail currents as follows: an atrial cell was depolarized from −80 mV and clamped for approximately 2 ms, or a duration not leading to channel inactivation, in 10 mV steps from −80 to +50 mV. After the conditioning prepulse to various voltages, the cell was clamped back to −60 mV and the tail currents elicited were measured. Steady-state activation was determined by comparing the tail currents elicited at each varying prepulse voltage with the maximum current elicited, yielding a normalized activation curve. The same protocol was performed in the absence and presence of T₃.

Voltage dependence of steady-state inactivation of I_Na was determined by first applying a depolarizing voltage step to maximally activate I_Na. Then, the cell was held at −120 mV and clamped in 10 mV increments to +20 mV for 1 s. Following this initial conditioning pulse, the cell was returned to −120 mV to 2 ms, after which the cell was clamped to −40 mV to activate I_Na. Inward currents, elicited by the test pulse to −40 mV, were normalized to that obtained in the absence of a preceding conditioning pulse and compared in the absence and presence of T₃. Data were normalized and fitted using a Boltzmann function: I_t/I_max = {1 + exp[(V_c – V_0.5)/k]}⁻¹, where V_c is the voltage of the conditioning pulse, V_0.5 is half-maximal activation or inactivation voltage and k is the slope factor.

The curve for recovery from inactivation was determined by holding the cell at −110 mV and clamping to −40 mV for 1 s. This initial conditioning pulse was followed by a variable time interval (from 0.2 ms to 150 ms) at −110 mV after which the cell was test clamped again to −40 mV for 50 ms. Test current was compared with the conditioning current (I_test/I_cond) at each recovery time to yield a normalized value.

Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using the fluorescent Ca²⁺ indicator indo-1, as previously described (Hüser et al. 2000b). Myocytes were loaded with Ca²⁺ indicator by exposure to 5 μm acetoxymethyl ester of indo-1 (indo-1 AM, Molecular Probes, Eugene, OR, USA) in 1 ml Tyrode solution containing 0.001 g ml⁻¹ of a Pluronic F-127 for 10 min at room temperature. Cells were then washed for 10 min to allow de-esterification of the indicator. A coverslip with the attached cells was mounted on the stage of an inverted microscope. For spatially averaged single cell [Ca²⁺]_i measurements indo-1 fluorescence was excited at 357 nm. Cellular fluorescence signals were recorded simultaneously at 405 nm (F₄₀₅) and 485 nm (F₄₈₅). Changes of [Ca²⁺]_i are expressed as changes in the ratio R = F₄₀₅/F₄₈₅. [Ca²⁺]_i transients were evoked by field stimulation using 3 ms duration suprathreshold voltage pulses delivered through a pair of extracellular platinum electrodes. [Ca²⁺]_i transient amplitude was measured as the difference between baseline and peak [Ca²⁺]_i. Cells were electrically stimulated at 1 Hz and experiments were performed at room temperature.

Data are expressed as mean ± s.e.m. and were statistically analysed for significance using paired and unpaired Student's t test at P < 0.05. The n value indicates the number of cells tested. Drugs and chemicals included: triiodo-l-thyronine (T₃); reverse T₃ (rT₃); l-thyroxine (T₄); ryanodine; verapamil; 4-aminopyridine (all from Sigma); tetrodotoxin (TTX) (Calbiochem). The procedures used in this study were in accordance with the guidelines of the Animal Care and Use Committee of Loyola University Medical Centre.

Results

Figure 1A shows superimposed traces of I_Na elicited by depolarizing clamp steps from −80 to −40 mV in the absence and presence of 10 nm T₃. Exposure to T₃ for approximately 5 min increased I_Na from −17 to −26 pA pF⁻¹. There were no discernable effects of T₃ on holding current or the time course of I_Na decay. Washout of T₃ returned I_Na to −20 pA pF⁻¹. In a total of seven cells studied, acute exposure to T₃ increased I_Na from −34 ± 7 to −45 ± 10 pA pF⁻¹ (+32 %; P < 0.05). Washout of T₃ for approximately 5 min returned I_Na to −34 ± 7 pA pF⁻¹. Panel B shows the effects of T₃ on the I_Na current-voltage (I-V) relationship. T₃ significantly increased I_Na at voltages from −40 to +20 mV without affecting the threshold of I_Na activation, the voltage at which peak I_Na was activated (-40 mV) or the reversal potential (n = 7). Although the effect of T₃ to increase I_Na was quite small at more positive voltages, paired statistical analysis indicated a significant difference between control and test conditions. Further experiments showed that acute exposure to 10 nm reverse T₃ (rT₃) or 10 nm T₄ had no effect on I_Na compared with control values throughout the voltage range of −80 to +60 mV (n = 3; data not shown).

Figure 1. Acute exposure (5 min) to 10 nm T₃ increases I_Na.

A, I_Na was elicited by depolarizing clamp steps from a holding potential of −80 mV to −40 mV. T₃ elicited a reversible increase in peak I_Na (+17 %). B, the current-voltage relationship shows that T₃ significantly increased I_Na at voltages between −40 and +20 mV without affecting peak voltage dependence of I_Na activation or reversal potential. The effects of T₃ were fully reversible.

Figure 2 shows that the inward current increased by 10 nm T₃ (+23 %) is completely blocked by 10 μm tetrodotoxin (TTX). A total of five cells showed similar results. Additionally, when the holding potential was shifted to −40 mV, a voltage which inactivates I_Na, depolarizing steps (in the presence of TTX) failed to elicit any inward current (data not shown). Together, these findings indicate that T₃ increases TTX-sensitive I_Na, and that the effect is specific to T₃.

Figure 2. Tetrodotoxin (TTX) blocks the inward current increased by T₃.

Acute exposure to 10 nm T₃ increased the inward current activated by depolarization from −80 to −40 mV. Exposure to 10 μm TTX in the presence of T₃ blocked all inward current.

Further experiments showed that the effect of T₃ to increase I_Na is not due to alterations in Na⁺ channel kinetics. Acute exposure to 10 nm T₃ had no effect on the time course of I_Na decay (inactivation) (see Fig. 1A and Fig. 2). Moreover, the steady-state voltage of half-inactivation was not different in the absence (-83.5 mV) or presence (-82.5 mV) of T₃(n = 10). The Boltzmann slope factors (k = 7) also were the same under both conditions. The threshold of activation (-60 mV) and maximum I_Na activation (-20 mV) were unchanged by T₃. Also, I_Na reached 90 % recovery from inactivation in approximately 25 ms under both conditions.

Chronic exposure to thyroid hormone increases I_Ca,L (Binah et al. 1987). We therefore examined the acute effects of T₃ on I_Ca,L. Figure 3 shows selected recordings of I_Ca,L in the absence (A) and presence (B) of 10 nm T₃ and the effect of T₃ on the I-V relationship of I_Ca,L (C). Activation of I_Ca,L from a holding potential of −40 mV inactivates Na⁺ channels and therefore eliminates any potential secondary effects of T₃ mediated via stimulation of I_Na. Acute exposure to T₃ (for up to 15 min) had no effect on I_Ca,L throughout the voltage range of activation (n = 5). At peak activation voltage (0 mV), I_Ca,L amplitude was not significantly different in the absence (-461 ± 54 pA) and presence (-468 ± 63 pA) of T₃.

Figure 3. Acute exposure to 10 nm T₃ has no effect on L-type Ca²⁺ current (I_Ca,L).

A, control I_Ca,L elicited by depolarizing clamp steps from −40 to 0 mV. B, T₃ had no effect on peak I_Ca,L or I_Ca,L inactivation. C, current-voltage relationship shows T₃ had no effect on voltage dependence of peak I_Ca,L activation or reversal potential.

The relatively negative voltage range of I_Na activation overlaps with the activation range of T-type Ca²⁺ current (I_Ca,T). We therefore examined whether the effects of T₃ to increase inward current could be mediated, in part, via I_Ca,T by testing the effects of T₃ in the absence and presence of 50 μm nickel, an effective blocker of I_Ca,T (Zhou & Lipsius, 1994). Maximal I_Na was activated by voltage steps from −80 to −40 mV. In a total of five cells tested, acute exposure to 10 nm T₃ elicited a typical increase in peak I_Na from a control value of −46 ± 2 to −55 ± 4 pA pF⁻¹. Addition of 50 μm nickel had no significant effect (-53 ± 3 pA pF⁻¹) on T₃-induced increases in I_Na (data not shown), suggesting that I_Ca,T was not responsible for the increase in inward current induced by T₃.

Stimulation of Na⁺ influx via I_Na can stimulate Ca²⁺ uptake via reverse-mode Na⁺-Ca²⁺ exchange and thereby load SR Ca²⁺ stores (Sipido et al. 1995). By this mechanism, T₃ would be expected to enhance cell shortening. To minimize intracellular dialysis and avoid buffering of intracellular Ca²⁺ we used a perforated patch recording method to record action potentials and trigger contractions. As shown in Fig. 4, action potentials (top) and cell shortening (bottom) were elicited by electrical stimulation at 1 Hz. Although acute exposure (5 min) to 10 nm T₃ did not significantly affect action potential configuration (action potential duration at 90 % repolarization: control; 353 ± 5 vs. T₃; 350 ± 12 ms; n = 4), T₃ prominently increased cell shortening (63 ± 15 %; n = 4). Note that the positive inotropic effects of T₃ were reversed within minutes of removing T₃ from the bath.

Figure 4. Acute exposure to 10 nm T₃ increases action potential-induced contraction.

A, control recording of atrial action potentials and contraction elicited by field stimulation at 1 Hz. B, T₃ had little effect on action potential configuration but significantly increased contraction amplitude. C, washout of T₃ from the tissue bath returned contraction amplitude toward control (recovery).

Acute exposure to T₃ also could elicit spontaneous activity arising from delayed after-depolarizations. Figure 5A shows a typical response where acute exposure of a stimulated (1 Hz) atrial cell to 10 nm T₃ led to the development of delayed after-depolarizations and spontaneous action potentials (asterisks). Similar results were obtained in 11 of 15 cells tested. Withdrawing T₃ abolished abnormal activity and restored control conditions. Figure 5B shows that after-depolarizations and spontaneous beats elicited by T₃ were abolished by exposure (2 min) to 10 μm ryanodine (Rya), an alkaloid that interferes with SR Ca²⁺ release. Similar results were obtained in a total of four cells studied. During exposure to T₃, stimulated action potential durations were variable, depending on whether the stimulated action potential was preceded by a spontaneous Ca²⁺-mediated depolarization. This is consistent with the idea that the configuration of each stimulated action potential was modulated by the amount of SR Ca²⁺ available for release and its effect of stimulating Na⁺-Ca²⁺ exchange current. The fact that ryanodine normalized action potential duration in the presence of T₃ supports this interpretation. These findings indicate that T₃ can induce overload of SR Ca²⁺ content, presumably by enhancing Na⁺ influx via I_Na.

Figure 5. Acute exposure to 10 nm T₃ can induce delayed after-depolarizations and spontaneous activity.

A, control recordings show action potential elicited by field stimulation at 1 Hz; T₃ induced delayed after-depolarizations that reached threshold to form spontaneous (non-driven) action potentials (*); washout of T₃ abolished abnormal activities and returned action potential configuration to control. B, control action potentials elicited by field stimulation at 1 Hz; T₃ induced delayed after-depolarizations and spontaneous beats (*); addition of 10 μm ryanodine (Rya) abolished abnormal activities and returned action potential configuration to control.

In the following experiments we used the Ca²⁺-sensitive fluorescent indicator, indo-1, to visualize the effects of T₃ on [Ca²⁺]_i. Figure 6A shows control [Ca²⁺]_i transients elicited by field stimulation at 1 Hz recorded from the same cell. Exposure to 10 nm T₃ for 5 min increased both baseline intracellular Ca²⁺ and peak Ca²⁺ transient amplitudes. After 10 min of exposure, the effects of T₃ reached steady-state with only small further increases in both baseline [Ca²⁺]_i and peak [Ca²⁺]_i transient amplitude. Note that within minutes of washing out T₃, baseline [Ca²⁺]_i and [Ca²⁺]_i transient amplitude returned to control levels. Similar experiments showed that 10 nm rT₃ had no effect on [Ca²⁺]_i (n = 4; data not shown). In a total of seven cells, at 5 min of exposure, T₃ significantly increased baseline [Ca²⁺]_i (control, 2.03 ± 0.05 vs. T₃, 2.32 ± 0.06; +14 %; P < 0.05) and [Ca²⁺]_i transient amplitude (control, 1.13 ± 0.13 vs. T₃, 2.11 ± 0.2; +87 %; P < 0.05). At 10 min of T₃ exposure, further increases in baseline [Ca²⁺]_i and [Ca²⁺]_i transient amplitude were not statistically significant, indicating that steady-state effects of T₃ were reached.

Figure 6. Acute exposure to 10 nm T₃ increases intracellular Ca²⁺ content and release.

A, control [Ca²⁺]_i transients elicited by field stimulation at 1 Hz. Exposure to T₃ for 5 min increased baseline [Ca²⁺]_i and [Ca²⁺]_i transient amplitude without affecting relaxation of [Ca²⁺]_i transients. After 10 min of exposure to T₃ there were further increases in both baseline [Ca²⁺]_i and [Ca²⁺]_i transient amplitude. Washout of T₃ restored control parameters. B, control recordings from another atrial cell show [Ca²⁺]_i transients followed by SR Ca²⁺ release induced by rapid exposure to10 mm caffeine. Acute exposure to 10 nm T₃ increased baseline [Ca²⁺]_i, [Ca²⁺]_i transient amplitude and SR Ca²⁺ release induced by 10 mm caffeine.

To determine whether T₃ increased SR Ca²⁺ content, single atrial myocytes were rapidly exposed to 10 mm caffeine to release SR Ca²⁺. Caffeine was tested in the same cell before (control) and during a 5 min exposure to 10 nm T₃. Cells were field-stimulated at 1 Hz for approximately 5 min before each caffeine exposure to ensure steady-state loading of SR Ca²⁺. The left trace of Fig. 6B shows control steady-state [Ca²⁺]_i transients elicited by field stimulation. Within approximately 5 s of switching off the stimulation, rapid exposure to 10 mm caffeine elicited a large [Ca²⁺]_i transient. The right trace of Fig. 6B shows that a 5 min exposure to T₃ increased the baseline [Ca²⁺]_i, the amplitude of the [Ca²⁺]_i transients and the caffeine-induced [Ca²⁺]_i transient compared with control. In a total of three cells, acute exposure to T₃ significantly increased the caffeine-induced [Ca²⁺]_i transient compared with control (control, 2.49 ± 0.05 vs. T₃, 3.39 ± 0.14; +36 %; P < 0.05). Together, these findings indicate that acute T₃ exposure increases intracellular SR Ca²⁺ content and release.

Discussion

The present results demonstrate that in atrial myocytes, acute exposure to T₃ increases TTX-sensitive I_Na. As a result of enhanced Na⁺ influx, T₃ indirectly increases intracellular Ca²⁺ via Na⁺-Ca²⁺ exchange, leading to positive inotropic effects and possible Ca²⁺-mediated dysrhythmias. The acute effects of T₃ on I_Na were achieved within 5 min of exposure, a time frame too short to be consistent with genomic effects. This relatively rapid response to T₃ is consistent with other studies in which acute exposure to T₃ elicited rapid membrane-mediated changes in transmembrane transport of ions and substrates (Segal, 1989). Moreover, as shown in the present study, the stimulatory effects of T₃ on I_Na, cell shortening, the development of arrhythmic activities and [Ca²⁺]_i transients were all reversible within minutes of removing T₃ from the tissue bath. Together, these findings support the idea that in the present experiments acute exposure to T₃ acted via non-genomic mechanisms.

T₃-induced increases in macroscopic I_Na in atrial myocytes are consistent with the effect of T₃ to increase single Na⁺ channel bursting behaviour in ventricular myocytes (Dudley & Baumgarten, 1993). In the present experiments, T₃ acutely increased I_Na by about 32 % within approximately 5 min. However, as a result of cell dialysis, ‘run down’ decreased I_Na by approximately 10 % over the same time period. Moreover, the effects of T₃ on I_Na were studied at room temperature and in one-third the normal extracellular Na⁺ concentration. Each of these factors would be expected to attenuate the absolute effects of T₃ on I_Na. It therefore seems likely that these experiments underestimate the actual stimulatory effect of T₃ on I_Na amplitude. This idea is supported by the present findings that T₃ more than doubled the stimulated [Ca²⁺]_i transient amplitude and cell shortening. Several additional factors may contribute to these prominent effects of T₃ on [Ca²⁺]_i. First, the effects of T₃ on [Ca²⁺]_i transients and cell shortening were tested in cells stimulated by action potentials in normal extracellular Na⁺ concentration. In addition, Ca²⁺ influx via reverse-mode Na⁺-Ca²⁺ exchange, especially following Na⁺ influx via I_Na, can contribute directly to triggering SR Ca²⁺ release (Leblanc & Hume, 1990; Lipp & Niggli, 1994; Lipp et al. 2002). Furthermore, an increase in SR Ca²⁺ content can increase the fractional release of intracellular Ca²⁺ (Lukyanenko et al. 1996; Sitsapesan & Williams, 1997; Xu & Meissner, 1998). These considerations suggest that the acute effects of T₃ may be quite prominent under physiological conditions. The present results also show that the increase in peak I_Na was specific to the actions of T₃. T₄ and rT3, other hormones produced by the thyroid gland, produced no effect on I_Na. This type of specificity has been reported for the acute effects of T₃ (but not rT₃) to increase the inward rectifier K⁺ current (I_k1) (Gotzsche, 1994).

The present results indicate that acute exposure to T₃ does not increase I_Na through alterations in the gating kinetics of I_Na activation, inactivation or recovery from inactivation. In ventricular myocytes, single channel recordings showed that acute T₃ exposure did not increase single Na⁺ channel current amplitude but rather increased the bursting behaviour of the channel, i.e. caused an increase in open probability (Dudley & Baumgarten, 1993). Similarly, in ventricular myocytes, acute T₃ exposure increases the open probability of I_k1 without affecting channel kinetics (Sakaguchi et al. 1996). Based on these considerations and the present data, it seems likely that T₃ exerts an increase in macroscopic Na⁺ current by increasing the open probability of the channel.

What is the mechanism by which T₃ rapidly increases I_Na? The answer to this question is not entirely clear. It is known that T₃ is taken up in neonatal cardiomyocytes by an energy-dependent carrier-mediated mechanism that may be partly dependent on the Na⁺ gradient (Everts et al. 2001). In addition, there are T₃ receptors in the cytoplasm that may function distinctly from their role in gene transcription in the nuclear compartment (Baumann et al. 2001), although their functional role is not known. However, Dudley & Baumgarten (1993) clearly showed that in rabbit ventricular myocytes, the acute effect of T₃ to increase Na⁺ channel activity is not mediated via second messenger signalling, and required that T₃ be in close proximity to the extracellular face of the channel. These findings suggest some sort of direct effect of T₃ on the channel and are consistent with the relatively rapid time course over which T₃ increases I_Na.

Several of the present results indicate that T₃ elevates [Ca²⁺]_i. T₃ increased cell shortening, and could elicit delayed after-depolarizations and spontaneous activity between stimulated beats, both manifestations of elevated SR Ca²⁺ content and release. Moreover, depletion of SR Ca²⁺ content and inhibition of SR Ca²⁺ release by ryanodine eliminated the arrhythmogenic effects of T₃. Also, direct measurements showed that T₃ increased caffeine-induced SR Ca²⁺ release, baseline [Ca²⁺]_i and [Ca²⁺]_i transient amplitude, consistent with elevated SR Ca²⁺ content and release. These findings are in agreement with the effects of acute T₃ exposure to increase tension-contractile activity in isolated muscle cells (Snow et al. 1992; Mager et al. 1992), isolated perfused hearts (Tielens et al. 1996), and the intact animal (Gotzsche, 1994). The fact that T₃ did not alter L-type Ca²⁺ current and that its effects on I_Na were unaltered by Ni²⁺, an inhibitor of I_Ca,T, suggests that the effects of T₃ to increase [Ca²⁺]_i are not mediated through Ca²⁺ influx via voltage-dependent Ca²⁺ channels. Moreover, the prominent effects of T₃ to increase cell shortening and [Ca²⁺]_i are not consistent with the modest effects of Ca²⁺ influx via I_Ca,T (Sipido et al. 1998; Zhou & January, 1998). Alternatively, increases in Na⁺ influx via I_Na can alter intracellular Na⁺ gradients and thereby reverse Na⁺-Ca²⁺ exchange, resulting in significant Ca²⁺ influx (Sipido et al. 1995). This mode of Ca²⁺ influx is consistent with the increase in baseline intracellular Ca²⁺ concentration (Lipp et al. 2002) and can account for the increased intracellular Ca²⁺ content and release induced by T₃.

The present results are consistent with clinical findings seen in hyperthyroid patients who traditionally present with classic symptoms of increased heart rate, stroke volume, cardiac output and cardiac contractility along with potential atrial irregularities including atrial fibrillation (Klein & Levey, 2000; Klein & Ojamaa, 2001). The present results, therefore, indicate that the non-genomic effects of T₃ may contribute substantially to the effects of thyroid hormone traditionally attributed to chronic, genomic mechanisms. With regard to pacemaker activity, pacemakers within the sinoatrial (SA) node (Lipsius & Vassalle, 1978) as well as right atrial pacemakers outside of the SA node (Rubenstein & Lipsius, 1989) are partially dependent on activation of TTX-sensitive I_Na. Therefore, these pacemaker cell types are potential targets for T₃ action. Moreover, our laboratory (Hüser et al. 2000a), as well as others (Rigg & Terrar, 1996; Bogdanov et al. 2001), has shown that intracellular Ca²⁺ release is a key component underlying atrial pacemaker and SA node activity. Specifically, voltage-dependent activation of I_Ca,T triggers local SR Ca²⁺ release, which in turn stimulates Na⁺-Ca²⁺ exchange current to depolarize the pacemaker potential toward threshold (Hüser et al. 2000a). In fact, preliminary recordings from cat atrial pacemaker cells indicate that acute T₃ exposure stimulates pacemaker activity via a TTX-sensitive mechanism (Y. G. Wang & S.L. Lipsius, unpublished observations). Furthermore, in rat neonatal atrial myocytes, T₃ stimulates pacemaker rate and Na⁺-Ca²⁺ exchange current (Sun et al. 2001). By increasing [Ca²⁺]_i (via stimulation of I_Na), acute effects of T₃ can be expected to enhance the normal Ca²⁺-mediated mechanisms underlying SA node and right atrial pacemaker activities, thereby contributing to the sinus (atrial) tachycardia induced by elevated serum levels of T₃ in patients with long-standing hyperthyroidism.