Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (Im) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of −87 ± 2 mV and −83.9 ± 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of Im around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of Im was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased Im at −120 mV from 4.3 pA/pF to 27 pA/pF with an EC50 of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of Im fourfold, shifted its reversal potential from −78 ± 3 to −84 ± 3 mV, and stabilized the resting membrane potential at −92 ± 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or Im in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K+ current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.
- teleost heart
- I K,ACh
- cholinergic modulation
- action potential
the resting membrane potential is a key parameter in cardiac cells that directly regulates the steady-state inactivation and recovery from inactivation of ionic channels (22, 26) and thus modulates excitability and the refractory period of cardiac myocytes (22). The resting membrane potential also plays a key role in the regulation of contraction by modulating the Na+/Ca2+ exchange activity and the sarcoplasmic reticulum calcium content (15, 16, 29, 30). The resting membrane potential, in turn, is determined primarily by the activity of potassium channels. In the mammalian heart, the resting potential is primarily determined by the inwardly rectifying potassium current (IK1), and this current has also been proposed to set the resting potential in teleost myocytes (3, 31). Several other currents have been shown to modulate the resting potential in mammalian cardiac myocytes (3, 8, 19, 22, 25). The ACh-activated IK (IK,ACh) has been shown to modulate the membrane potential in mammalian atrial and, to a lesser extent, ventricular cells (19). This current has also been proposed to modulate the membrane potential in the teleost heart in which a basal cholinergic tone is present (12, 20, 21). The presence and potential role of IK,ACh has, however, not been investigated in the teleost heart. In contrast, some studies have used the current-clamp technique to activate contraction in trout atrial myocytes. The resting membrane potential recorded with the current-clamp technique in these studies was between −40 and −50 mV (27, 28). Considering that a resting potential between −40 and −50 mV among other things would lead to an almost complete steady-state inactivation of the Na+ current (11) and increased calcium loading of the sarcoplasmic reticulum (14) in the trout heart, the physiological relevance of such experimental conditions is not clear. The aim of the present study was, therefore, to compare the resting membrane potential in trout cardiac tissue and isolated myocytes and to characterize the ionic currents that set the resting membrane potential in atrial and ventricular myocytes. Our results show that trout ventricular myocytes have a robust IK (i.e., IK1) and a stable membrane potential. In contrast, at hyperpolarized potentials, the slope conductance of IK1 was sevenfold smaller in trout atrial cells, and application of current clamp in these cells resulted in unstable membrane potentials. ACh caused a marked stimulation of an inwardly rectifying current in atrial myocytes and stabilized the membrane potential, suggesting that this current may play an important role in the modulation of the membrane potential in the trout atrium.
Rainbow trout were obtained from a commercial trout farm and kept in tanks at 16°C with a 12:12-h light-dark photo period. Cardiac myocytes were isolated by enzymatic digestion of the trout heart as previously described (17). The myocte isolation procedure was approved by the ethical committee at the Universitat Autònoma de Barcelona and adheres to American Physiological Society's “Guiding Principles in the Care and Use of Animals” (1).
Membrane potentials were measured in trout atrial tissue preparations as previously described for trout ventricular tissue (13) with microelectrodes having a resistance of 30–50 MΩ. Action potentials (APs) were elicited by electrical field stimulation of the atrial tissue at a frequency of 0.2 Hz. Electrical potentials were recorded at a rate of 200 Hz with a data acquisition system (model MP100; Biopac Systems, Goleta CA).
Whole membrane current (Im) and APs were measured in isolated myocytes by using the perforated patch configuration with 250 μg/ml amphotericin B in the patch pipette. Pipette resistances varied between 1.5 and 3 MΩ, and the seal resistance immediately after seal formation was > 2 GΩ for all cells. Experiments were begun when the access resistance had decreased to less than four times the pipette resistance. The resting membrane potential was measured in the current-clamp configuration with the holding current set to 0 pA unless otherwise stated. APs were elicited every 2 s by a brief current injection (5 nA for 2 ms) and measured by holding the current at 0 pA. For measurements of Im, a holding potential of −80 mV was routinely used. To investigate the stimulatory effect of ACh on IK1, a 100-ms hyperpolarization from −80 to −120 mV every 2 s was used. The amplitude of the K+ current was measured as the current at the end of the hyperpolarization. To determine the current-voltage relationship, the membrane potential was switched from −80 mV to different test potentials for 200 ms, and the current amplitude at the end of the 200-ms pulse was measured. Solutions containing ACh and/or atropine were prepared immediately before the beginning of experiments. The intracellular solution had the following composition (in mM): 109.2 dl-aspartic acid (potassium salt), 46.8 KCl, 1 MgCl2, 3 MgATP, 5 Na2-phosphocreatine, 0.42 Li guanosine triphosphate (LiGTP), 10 HEPES. pH was adjusted to 7.2 with KOH at 20°C. Amphotericin B (250 μg/ml) was added to the intracellular medium from a 50 mg/ml stock solution. For the ruptured patch configuration, 10 mM EGTA was included in the pipette solution and amphotericin B was not included. Unless otherwise stated, the extracellular solution contained (in mM) 132 NaCl, 2.5 KCl, 4 NaHCO3, 0.33 NaH2PO4, 2 CaCl2, 1.6 MgCl2, 10 HEPES, 5 glucose, and 5 pyruvic acid. pH was adjusted to 7.5 with NaOH, at 20°C and 7.6 in experiments performed at 15°C.
Values are expressed as means ± SE. Data sets were tested for normality and statistical significance was determined with Student's t-test for paired comparisons or ANOVA for multiple comparisons. Values from each animal were averaged and, unless otherwise stated, n refers to the number of animals used in each experimental series.
AP and IK characteristics in trout cardiac myocytes.
Figure 1A shows representative AP recordings in trout atrial tissue, a trout atrial myocyte and a trout ventricular myocyte. In atrial tissue, the resting membrane potential was stable at −87 ± 2 mV in all stimulated preparations. The membrane potential was also stable in six unstimulated preparations but significantly more positive (−79 ± 2 mV, P < 0.02, n = 6).
In contrast, 50 out of 59 atrial myocytes had an unstable depolarized membrane potential near −45 mV when current was clamped at 0 pA, whereas the remaining cells had more stable membrane potentials near −88 mV. The resting potential was very stable in all ventricular cells examined. Interestingly, only a small change in the holding current of 5 or 10 pA had a profound effect on the resting membrane potential in the atrial myocytes, whereas the membrane potential of ventricular cells was unaffected by such a change in the holding current. Figure 1B shows representative atrial APs elicited at three different holding currents, and Table 1 summarizes the characteristics of atrial and ventricular myocytes as well as the AP characteristics of multicellular atrial tissue preparations. Table 1 also shows the AP characteristics in multicellular preparations at 15°C and with 4 mM extracellular potassium concentration, conditions that are commonly used to mimic physiological conditions. As expected, lowering of the experimental temperature to 15°C significantly slowed the kinetics of the AP, whereas no significant effect on the resting potential was observed.
To determine whether the instability of the atrial membrane potential is related to the amplitude of Im that set the membrane potential, the current-voltage relationship was established by using the perforated patch configuration. Figure 1C shows the whole Im recorded at different test potentials and the corresponding current-voltage relationships for 33 ventricular myocytes from seven trout and for 37 atrial myocytes from 14 trout are shown in Fig. 1D. The slope conductance of the whole Im between −140 and −80 mV was 65 ± 18 pS/pF for atrial cells and 443 ± 73 pS/pF for ventricular myocytes. Table 1 gives the average cell size, and reversal potential, and slope conductance of Im between −140 and −80 mV for atrial and ventricular myocytes.
IK,ACh in trout cardiac myocytes.
As an IK,ACh has been reported in mammalian and carp atrial myocytes (18, 32, 33), we hypothesized that the presence of an IK,ACh in the trout atrium may contribute to stabilize the membrane potential. To test this hypothesis, we first examined the effect of ACh on Im in isolated trout atrial myocytes. Figure 2 shows the effect of increasing doses of ACh on the Im elicited by hyperpolarizing the myocytes from −80 to −120 mV. Figure 2A shows representative currents before exposure to ACh and in the presence of increasing ACh concentration ([ACh]). As shown in Fig. 2B, ACh very potently and reversibly stimulated the inward current at −120 mV, and Fig. 2C shows the dose-response curve for ACh. Fitting data points from seven trout with a hyperbolic equation gave a basal current density of 4.3 pA/pF, an EC50 of 45 nM, and a maximally stimulated current of 27 pA/pF or about a sixfold stimulation of the basal current. Figure 2D compares the current-voltage relationship without ACh and with different ACh concentrations. The slope conductance between −80 and −140 mV increased from 62 ± 17 in the absence of ACh to 677 ± 137 pS/pF with 30 nM ACh. More importantly, the inward Im recorded at test potentials of −80 and −60 mV in the absence of ACh was shifted to outward currents by all the ACh concentrations employed. Figure 2E compares the effect of 3 nM ACh on the current-voltage relationship for the whole Im recorded in the perforated patch configuration and for the IK recorded in the ruptured patch configuration with 10 mM EGTA in the patch pipette and 1 μM tetrodotoxin plus 3 μM nifedipine in the bath solution. The voltage dependency for the inwardly rectifying current was comparable for the two patch configurations.
To directly examine the effect of ACh on the membrane potential, atrial myocytes were exposed to increasing concentrations of ACh in the current-clamp configuration. Figure 3A shows representative AP-traces from an atrial myocyte. Notice that a low [ACh] of 3 nM stabilized the resting membrane potential at −96 mV even though this [ACh] only had a modest effect on the K+-current as shown in the corresponding current traces in Fig. 3B. As seen in Fig. 3A, ACh also reduced the AP duration (APD) at 90% repolarization (APD90) and the time to 25% repolarization (APD25). Importantly, 3 nM ACh also shifted the membrane potential to −91 ± 4 mV in four trout with a depolarized membrane potential. Figure 3C shows that the stimulatory effect was overcome by the muscarinic M2-receptor antagonist atropine, thus confirming the specificity of the ACh effect. Table 2 shows that this was also true when measuring the effect of ACh on membrane potential in field-stimulated atrial tissue preparations, and it summarizes the effect of 3 nM ACh on AP-parameters and resting membrane potential, as well as the reversal potential and the slope conductance of Im between −80 and −140 mV in isolated atrial myocytes. In accordance with these results, ACh also induced a 1.9 ± 0.7 mV hyperpolarization that was reversed by atropine in five unstimulated tissue preparations. Thus, ACh induced an average hyperpolarization of 2.8 ± 0.8 mV in the 13 tissue preparations examined (P < 0.01), which was reversed by atropine (2.7 ± 0.7 mV depolarization, P < 0.01).
ACh-dependent regulation of AP characteristics.
To determine whether the observed differences in the resting membrane potentials recorded in atrial tissue and myocytes could at least partly be accounted for by an ACh tone in the trout atrial tissue preparations, we compared the effect of atropine in trout atrial tissue and myocytes. Figure 4A shows recordings of APs before and after exposure of a multicellular atrial preparation to 1 μM atropine. Atropine depolarized the membrane potential in some but not all cells and significantly prolonged the APD90 (P < 0.02, n = 5). In isolated myocytes, atropine did not alter AP characteristics or the resting membrane potential in 10 atrial myocytes that all had a depolarized membrane potential, nor did it change the characteristics of the Im in isolated atrial myocytes (Fig. 4B). At −120 mV, the Im was 2.61 ± 0.23 and 2.67 ± 0.28 pA/pF (P = 0.87, n = 6) with and without atropine, respectively. Moreover, atropine at a concentration of 1 μM did not affect the current-voltage relationship as shown in Fig. 4C. When using the ruptured patch configuration with EGTA in the patch pipette and tetrodotoxin plus nifedipine in the bath solution, 100 nM atropine was also without effect on the current-voltage relationship (data not shown).
Repolarization of the AP is brought about by potassium efflux from the cardiac myocytes through ionic channels, and it is essential to assure that ion channels can recover from voltage-dependent inactivation. It also favors calcium removal from the cytosol by forward mode electrogenic Na+/Ca2+ exchange and, thus, relaxation of contraction. The role of specific potassium channels in the repolarization and maintenance of the resting membrane potential has been extensively studied in mammalian cardiac myocytes (3, 5, 8, 18, 24). However, the mechanisms regulating repolarization and the maintenance of the resting membrane potential in the fish heart needs clarification. Thus, available literature reports resting membrane potentials near −45 mV in trout atrial myocytes, and maintenance of this potential is attributed to the IK1 and the rapidly activating delayed rectifying IK (31). Since a resting membrane potential of −45 mV would prevent atrial excitability, the use of the current-clamp technique to record membrane potentials is unsuitable for trout atrial myocytes and/or other potassium currents have to be operating in atrial myocytes in situ.
In 1984, Noma et al., (24) proposed that ACh-activated K+-channels, when activated by neural stimulation, may play a major role in generating a resting K+ conductance in mammalian cardiac nodal cells. Furthermore, pharmacological manipulations of heart rate and heart rate variability in the teleost heart (1a, 20) support the presence of a basal cholinergic tone in the trout heart (20). We, therefore, hypothesized that ACh-activated K+ channels may also play an important role in the modulation of the resting membrane potential and AP characteristics in the trout atrium.
AP characteristics in trout heart.
To test this hypothesis, we first compared the characteristics of APs recorded in trout atrial and ventricular myocytes. Our measurements in trout ventricular myocytes were similar to previously reported values in myocytes (10, 31) and tissue (13) confirming that the current-clamp technique can faithfully record membrane potentials in trout ventricular myocytes under our experimental conditions. In contrast, 50 out of 59 atrial myocytes had an unstable depolarized membrane potential, whereas only 9 out of 59 cells had APs with characteristics similar to those measured in atrial tissue from 11 trout. The unstable membrane potential was associated with a sevenfold smaller slope conductance of the Im in atrial rather than ventricular myocytes. This is similar to previous reports that have investigated IK in fish and mammalian cardiac myocytes (3, 18, 19, 31, 32). Interestingly, the IK density in carp atrial myocytes at −120 mV is about twice the IK density measured in trout atrial myocytes, and this difference may explain why stable APs with a resting potential near −80 mV can be recorded in carp but not in trout atrial myocytes.
The smaller Im density and slope conductance near the resting membrane potential in trout atrial myocytes also explains the strong influence of the holding current on the membrane potential.
Atrial myocytes vs. atrial tissue.
Previous recordings of APs in the current-clamp configuration, used for AP clamping in trout atrial myocytes, have reported resting membrane potentials around −45 mV (28), which is similar to our measurements in the majority of cells examined. This is, however, a 40-mV depolarization compared with our measurements in atrial tissue with 2.5 mM extracellular K+, a plasma K+ concentration ([K+]) measured in nonexercising trout (23). During exercise, the plasma [K+] has been reported to rise to 4 mM (23), a value closer to that used in previous AP measurements in trout atrial myocytes. With 4 mM extracellular K+ and a bath temperature of 15°C, trout atrial AP showed significantly slower kinetics, but the resting membrane potential was not significantly changed under these conditions (see Table 1).
Since a resting membrane potential of −45 mV would inactivate Na+-channels and prevent excitability, we considered three possible reasons for the divergence between our AP recordings in trout atrial tissue and isolated myocytes: 1) inability of the current-clamp technique to faithfully record APs in trout atrial myocytes; 2) partial digestion of potassium channels by the myocyte isolation procedure; and 3) presence of a cholinergic tone in the trout atrial tissue under our experimental conditions.
With respect to the first possibility, the small Im density and slope conductance near the resting membrane potential certainly make trout atrial myocytes much more sensitive to leakage current. Indeed, clamping the current to 0 pA for AP recordings may not represent a negligible error in trout atrial myocytes, even when the leakage current is small. Thus, our results show that an error of 5–10 pA in the holding current has a dramatic effect on the resting membrane potential in atrial but not ventricular myocytes. Therefore, variations in the leakage current among cells could also influence the measured resting membrane potential. While only cells with a seal resistance >2 GΩ were used for experimentation, we cannot rule out that small differences in leakage current may at least partly account for the large variability in the resting membrane potential in isolated atrial myocytes.
The small Im density may in turn result from a partial digestion of the K+-channels by the enzymes used for myocyte isolation. We cannot rule out this possibility, but the observed tendency toward a larger slope conductance in cells with a resting potential near −80 mV than in those with a depolarized membrane potential was not significant (see Table 2). Given that IK,ACh has been reported in atrial myocyte preparations from other species (7, 19, 32, 33), we considered the possibility that the presence of a basal cholinergic tone in the trout atrium could contribute to stabilize and hyperpolarize the resting membrane potential.
IK,ACh in trout cardiac myocytes.
To test whether a cholinergic tone could modulate the resting membrane potential and its stability, we investigated the effect of pharmacological manipulation of the cholinergic stimulus in trout atrial myocytes and tissue. Our findings show that even with a modest ACh concentration of 3 nM, the steady-state Im amplitude at −120 mV [Im(−120)] is doubled and that this is sufficient to stabilize the resting membrane potential at −92 ± 4 mV. Current recordings in the ruptured patch configuration with 10 mM EGTA in the patch pipette and 1 μM tetrodotoxin plus 3 μM nifedipine in the patch pipette to eliminate Na+/Ca2+ exchange, sodium and L-type calcium currents confirmed that ACh stimulates an IK1. Higher concentrations of ACh strongly stimulate Im in trout atrial myocytes with an EC50 of 45 nM and a maximally stimulated current of 27 pA/pF. The EC50 is somewhat lower than values reported in other studies (4, 6, 9, 19) and may partially account for the potent effect of modest ACh concentrations on the resting membrane potential. However, the low density of other potassium currents in the trout atrium also makes the relative contribution of the IK,ACh large even though the increase in the current amplitude induced by 3 nM ACh is modest. Higher concentrations of ACh led to a further stimulation of Im, hyperpolarization of the resting membrane potential, and a strong reduction of the APD. As a consequence, APs were extremely short and the amplitude was reduced in the presence of 10 nM ACh, and with 30 nM ACh APs could not be elicited in any of the cells examined. The observed biphasic response to ACh with an initial peak followed by a relaxation of the stimulatory effect to a new steady-state level is in accordance with data from mammalian atrium where relaxation of the stimulatory effect of ACh is ascribed to desensitization of the ACh-activated K+ channel (2, 6).
The prolongation of the APD by atropine in trout atrial tissue, but not in atrial myocytes suggests the presence of a cholinergic tone in our atrial tissue preparations. Although a cholinergic tone is not expected in isolated tissue preparations, one explanation for our observations would be that field stimulation of the atrial tissue stimulates ACh release from nerve endings, thus creating an artificial cholinergic tone. In accordance with this, the resting membrane potential was more depolarized in resting atrial preparations than in preparations subjected to field stimulation.
Although we cannot compare this artificial cholinergic tone to the settings in vivo, these results show that a cholinergic tone could modulate the membrane potential and its stability. Moreover, the small slope conductance of Im near the resting potential results in a significant impact of low ACh concentrations on the membrane potential and its stability. This, combined with reports suggesting the presence of a basal cholinergic tone in the teleost and the trout heart (12, 20, 21) makes it conceivable that ACh contributes to the control and stability of the resting membrane potential and the APD in the trout atrium. This, in turn, modulates excitability and refractoriness of the atrial tissue and thereby also the regulation of intracellular calcium handling (3, 14).
In summary, we have shown that trout atrial, but not ventricular, cells are highly sensitive to the holding current when current clamped. In accordance with this, a robust IK1 is present in trout ventricular, but not atrial, myocytes. In contrast, an IK,ACh is present in trout atrial, but not ventricular, myocytes. Stimulation of this current stabilizes the resting membrane potential in atrial myocytes. Together, the results suggest that this current may play an important role in the modulation of the resting membrane potential and the APD. These results have important consequences for measurements of the membrane potential with current-clamp technique in myocytes with a small current density around the resting membrane potential, and call for a determination of the cholinergic tone in the trout atrium.
This work was supported by a Ramon y Cajal Grant (to L. Hove-Madsen), the Danish Research Council, and Grant SGR-0205 from the Departament d’Universitats Reserca i Societat de la Informació (Generalitat de Catalunya).
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