Background: Action potential (AP) contours vary considerably between normal adult and aged right atrial fibers. The ionic bases for these differences remain unknown. Therefore we studied Ca2+ and K+ currents in cells from adult and aged canine right atria (RA). Methods and Results: We used whole cell patch clamp recording techniques to measure L-type Ca2+ currents (ICaL) with either Ca2+ or Ba2+ (3 mM) as the charge carrier, and both the transient outward (Ito) and sustained potassium currents (Isus) in cells dispersed from normal adult (Adult, 2–5 years) and older dogs (Aged, >8 years). There is a significant reduction in peak ICaL (47%) and IBaL (43%) in Aged cells, yet differences in IBaL disappear with maximal β adrenergic stimulation (isoproterenol, 1 μM). Composite Ito and Isus densities were significantly increased in the Aged versus Adult cell group (by 31 and 27% at +50 mV, respectively). Ito decay during a maintained depolarization was slowed in Aged cells. Furthermore, Ito steady-state inactivation curve was shifted positively in Aged cells. Finally, composite Ito and Isus currents of Aged cells were more sensitive to tetraethylammonium chloride (TEA), a specific inhibitor of some types of K+ currents. In the presence of TEA (5 mM), Ito in Aged cells was significantly greater than that in Adult cells. Conclusions: Ionic currents differ in Aged versus Adult right atrial cells, such that a reduced Ca2+ current and augmented outward currents could contribute significantly to the altered AP contour of the Aged RA cell. Adrenergic stimulation appears to restore Ba2+ currents in Aged cells. Finally, an augmented TEA sensitive current plays a role in changes of Isus in Aged right atrial cells.
Time for primary review 27 days.
Atrial fibrillation is a major public health problem in the US [1,2] afflicting ∼5% of people aged >65 years [3–6]. While several human and experimental studies have defined the changes in cellular and ionic properties of cells from fibrillating and non-fibrillating atria [7–14], few have defined the changes in ionic currents that determine the action potential (AP) of cells from aging atria. We have previously shown  that, with age, the action potential plateau is increasingly negative, APD30 decreases and APD90 increases. This is true for cellular action potentials recorded from Bachman's Bundle and from right atria (RA) pectinate muscle. Further, nisoldipine suppresses the plateau level in cells from normal adult dogs (Adult, 2–5 years) more than in cells from older dogs (Aged, >8 years). Since the action potential plateau is determined by a balance of inward and outward currents, we hypothesized that changes in both the L-type Ca2+ current and K+ currents important to atrial repolarization may underlie the alterations in action potential contour.
2.1 Animal preparation
This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, 1996).
Dogs were anesthetized with pentobarbital, 30 mg/kg i.v., and hearts removed via a thoracotomy. A section of RA freewall was excised for myocyte studies to minimize the heterogeneity in ion channel function reported for normal canine atria . RA pectinates were removed from adjacent tissue for study of cellular electrophysiological properties. Action potential data have been reported by Anyukhovsky et al. .
Two groups of mongrel dogs (16–22 kg) were studied, adults (2–5 years, Adult) and old (>8 years, Aged). As in the companion study, ages were estimated during physical examination. These Aged dogs, while all in normal sinus rhythm, had increased P wave duration .
2.2 Myocyte preparation
Single calcium tolerant atrial cells were dispersed using a modification of our previously described method . Briefly, the tissue was rinsed twice in a Ca2+-free solution (in mM: NaCl, 115; KCl, 5; sucrose, 35; dextrose, 10; HEPES, 10; taurine, 4; pH 6.95) and triturated in 20 ml of enzyme-containing solution (collagenase-II from Worthington Biochemical, 0.13 mg/ml; 36–37°C) for 30 min. This solution was then decanted and discarded. The second trituration was discarded after 30 min. The next six to seven triturations were each done for 15 min. Each time the solution was centrifuged at 500 rpm for 3 min to collect the supernatant and dispersed cells. Resuspension solution was changed every 30 min for solutions containing increasing concentrations of Ca2+ (0–0.5 mM). The living atrial cell yield was ∼30–40%. Only rod-shaped cells with staircase ends, clear cross striations and surface membranes free from blebs were studied.
2.3 Experimental conditions
2.3.1 Calcium/barium current studies
Cells were initially superfused with normal Tyrode's solution. Patch pipette resistances equaled 1–2 MΩ when filled with the following solution (in mM): CsOH 125, aspartic acid 125, TEA 20, HEPES 10, Mg-ATP 5, EGTA 10, creatine phosphate 3.6, pH 7.3 with CsOH. After gigaohm seal formation and membrane rupture, 5–10 min were allowed for intracellular dialysis before switching to a Na+- and K+-free solution containing (in mM): CaCl2 3, TEA 140, MgCl2 0.5, dextrose 10, HEPES 12, pH 7.3 with CsOH, 35°C . In all experiments, 2 mM 4-aminopyridine and ryanodine (2 μM) were added to the external solution to block current flow through voltage-dependent transient outward K+ channel (Ito) and to inhibit Ca2+ release from the SR. ICaL were determined as before  and magnitudes were normalized by each cell's membrane capacitance (pF) and expressed as current density (pA/pF). Cell capacitance averaged 62.5±3.8 pF in Adult cells (n = 35) and 76.2±3.3 pF in Aged cells (n = 53) (P<0.01). Voltages were not corrected for the liquid junction potential (∼10 mV). When myocytes are dialyzed during whole cell recordings, there is ‘rundown’ of peak ICaL with time [18,19]. In this study, we started data acquisition at similar times after membrane rupture since we determined that rundown was similar in all cells. Time course of ICaL decay, steady-state inactivation variables of peak and time course of recovery from inactivation of ICaL were determined as before . To directly compare Ca2+ channel activity, we selected recording conditions (both external and internal pipette solutions) so as to record Ca2+ channel activity when there are no other possible contaminating currents (e.g. NaCa currents and SR Ca2+ release activity). Thus we have used 3 mM Cao and 0Na, 0K solutions in our first set of experiments. In the internal pipette solution 10 mM EGTA was used to chelate Ca2+. Under these recording conditions, cells in both groups had similar currents (see Results) but we had concerns that EGTA was not a fast enough buffer to chelate Ca2+ ions in the very small space between the Ca2+ channel pore and subsarcolemmal SR (∼50 nm). Thus Ca2+ currents in atrial cells under these conditions would most likely be altered by marked Ca2+ induced inactivation of the channel.
Therefore in the next series of studies we used 3 mM Cao and BAPTA as the internal solution Ca2+ chelator . However, even in the presence of BAPTA, if Ca2+ is the charge carrier there can still be significant Ca2+ dependent inactivation of the L-type Ca2+ channel activity. Therefore in the final series of studies, we used Ba-BAPTA recording conditions. When Ba2+ is used as the charge carrier, there is less Ca2+-dependent inactivation of the channel than when Ca2+ is the charge carrier. Peak IBaL was taken as the current difference between peak and zero currents.
2.3.2 Potassium current studies
Patch pipette resistances equaled 1–2 MΩ when filled with the internal solution (in mM): KCl 140, MgCl2 1, EGTA 10, Mg-ATP 5, creatine phosphate 5, GTP 0.2, and HEPES 10, pH 7.2 with KOH. To measure outward currents, cells were externally superfused with a Na+-free solution (mM): N-methyl-d-glucamine (NMDG)-Cl 144, KCl 5.4, MgCl2 1, CaCl2 2.5, CdCl2 0.5 and HEPES 10, pH 7.4, 30–31°C. Na+ currents were suppressed via use of a Na+-free solution. ICaL was blocked with 0.5 mM Cd2+. Membrane currents associated with Na/Ca2+ exchange were eliminated by the absence of external Na+. Currents were elicited by 210-ms voltage step to Vt −50 and +60 mV from a holding potential of −60 mV at 0.1 Hz after a 10-ms prepulse to −90 mV to maximally activate Ito current. Ito amplitude was determined as the difference between the peak current and that at the end of the pulse (Fig. 5) or as stated (Fig. 6C). Isus was taken as the amplitude of the current at the end of test pulse relative to zero-current level. Ito decay was fit with a double exponential function to estimate time constants of current decay. ‘Steady-state’ inactivation relationships were determined using the following protocol: a 500-ms prepulse to various conditioning potentials (Vc) between −90 and +20 mV followed by a 210-ms test pulse to 60 mV. Peak current at each test pulse was expressed as a fraction of the current at Vc=−90 mV. Boltzmann equation was used to fit normalized data to obtain the half-maximal voltage (V0.5) and slope factor (k) for each cell. The time course of recovery from inactivation was evaluated with a paired-pulse protocol; two identical 210-ms pulses from a holding potential of −80 to +40 mV were delivered with increasing interpulse coupling intervals (IPI) from 5 to 5000 ms. The degree of recovery was determined by normalizing Ito at each IPI by the Ito at IPI 5000 ms. The time course of recovery was estimated by fitting the data points to a biexponential function using a simplex algorithm.
Isoproterenol (ISO) containing solutions were made on the day of the experiment from stock solution (1 mg/ml). Tetraethylammonium chloride (TEA; Sigma) was dissolved in ddH2O for stock solution.
Data are presented as mean±S.E.M. All data were tested using ANOVA for multiple comparisons. If significant changes occurred, group means were compared using Bonferroni's method. P<0.05 was considered significant.
3.1 Calcium/barium currents
The depressed plateau of action potentials of Aged RA cells (Fig. 1D) may be due to a reduced availability and/or kinetics of L-type Ca2+ currents. When ICaL were studied and compared under 3 mM Ca2+, 10 mM EGTAi conditions, we found no differences in Ca2+ current densities or kinetics (data not shown) at all test voltages in Adult and Aged groups (Fig. 1). However, we suspected there was pronounced Ca2+ dependent inactivation of ICaL under these conditions. Therefore, in another subset of cells where Ca2+ was charge carrier, we used the more rapidly acting chelator BAPTA (10 mM). Under these conditions, ICaL density was reduced in the Aged versus the Adult cell group at several test voltages (P<0.05) (Fig. 2). Further, when Ba2+ was the charge carrier, there remained a significant reduction in peak Ba2+ currents in the Aged versus the Adult cell groups as well as an acceleration of current decay (Fig. 3). This current reduction in Aged cells was unaccompanied by a significant change in availability of IBaL or its recovery from inactivation (Table 1,A).
|Group||Steady-state availability||Recovery from inactivation|
|V0.5 (mV)||k (mV)||IBaL (pA/pF)||n||τf (ms)||τs (ms)|
|V0.5 (mV)||k (mV)||Itomax (pA/pF)||n||τf (ms)||τs (ms)||n|
|Group||Steady-state availability||Recovery from inactivation|
|V0.5 (mV)||k (mV)||IBaL (pA/pF)||n||τf (ms)||τs (ms)|
|V0.5 (mV)||k (mV)||Itomax (pA/pF)||n||τf (ms)||τs (ms)||n|
Mean±S.E.M. V0.5 and k are average values of voltage at half maximal availability and slope factor as determined using a Boltzmann equation. Current densities for IBaL and Itomax at maximal voltage (−90 mV) are shown. τf and τs are time constants of recovery from inactivation. n is the number of cells.
Thus, Aged RA cells have lower peak Ca2+ and Ba2+ current densities than Adult RA cells. We next enhanced Ca2+ channel activity by promoting its phosphorylation using a short exposure (3 min) to a maximal concentration of isoproterenol (ISO; 1 μM). Under these recording conditions (Ba2+/BAPTAi) ISO produced a 1.3-fold increase in peak currents in Adult cells (Fig. 4). In contrast, ISO produced a significantly greater fold effect in Aged cells (Fig. 4C). However, the maximal ISO-stimulated current obtained in Aged cells was no different from that in Adult cells (Fig. 4D). Thus while Aged RA cells have a reduced function of basal Ca2+ channels, maximal β adrenergic stimulation of IBaL in Aged cells restores ‘normal’ Ca2+ channel function as evidenced in peak current recordings.
3.2 Potassium currents
Fig. 5A,B shows representative tracings of composite Ito and Isus from an Adult and Aged RA cell, respectively. Compared to Adult, the Aged cell shows a slowly decaying Ito and large Isus. Average current–voltage relations for Ito and Isus in Adult and Aged cell groups (Fig. 5C,D) show that Ito and Isus densities are significantly increased in the Aged group, and currents decay more slowly. For example, the fast time constant (τf) of decay of Ito at +50 mV was 9.9±0.1 ms in Adult (n = 46) and 12.4±0.1 ms in Aged cells (n = 42) (P<0.05) while the slow time constant (τs) of decay of composite Ito was 47.1±0.5 ms in Adult and 58.8±0.7 ms in Aged cells (P = 0.049).
A change in voltage-dependence and/or kinetic properties could account for the differences in K+ current densities in the Aged group. Accordingly we determined that the average ‘steady-state’ inactivation of composite Ito in Aged cells was shifted to more positive voltages than that in Adult RA cells (Table 1,B). This was accompanied by a slowing in recovery from inactivation of Ito in the Aged versus Adult group (Table 1,B).
To better understand the mechanisms of the changes in outward currents in Aged RA cells, we examined the effects of TEA on K+ currents in the two cell groups. Fig. 6 shows the different responses to TEA (5 mM) between an Adult and Aged RA cell. TEA sensitive current densities were greater in Aged versus Adult at Vt>+30 mV (Fig. 6C). Further, even in the presence of TEA, peak Ito current density in Aged RA cells remained significantly greater than that in Adult cells (e.g. at +50 mV, TEA insensitive Ito in Aged cells (n = 19) was 17.1±3.5 versus 9.2±1.2 pA/pF in Adult cells (n = 14)) while current decay and V0.5 of inactivation relations no longer differed between cells in the two groups. Thus with aging, RA cells show an augmentation of outward currents which are TEA sensitive.
We have shown that aging is associated with an attenuation of Ca2+ current function and an augmentation of K+ currents in right atrial cells.
4.1 Ca2+ currents of Aged atrial myocardium
There have been numerous reports of age-dependent changes in whole cell L-type Ca2+ currents in ventricular cells [21–24]. Recent data from rat ventricle suggest that single Ca2+ channel activity in cell-attached patches increases with age due to an increased number of active channels per patch as well as an increase of Po and availability . To our knowledge there are no comparable Ca2+ current studies documenting the effects of age on rat or human atrial cell currents.
We observed a significant reduction in the functionally available Ca2+ channels in Aged cells when intracellular Ca2+ was quickly chelated with BAPTA. Further, significant differences in current densities persisted when Ba2+ was the charge carrier suggesting that the basal level of available channels is reduced. Interestingly, differences in peak Ba2+ current densities between Aged and Adult cells disappeared with maximal β adrenergic stimulation. Further isoproterenol produced a greater stimulation of IBaL in Aged versus Adult cells.
It is generally believed that adrenergic responsiveness diminishes with age  since Ca2+ currents of Aged ventricle appear ‘unresponsive’ to β adrenergic stimulation, perhaps due to an altered basal activity of each channel . From our studies, the atrial Ca2+ channel and its adrenergic control respond differently with age. Isoproterenol elicits a robust response in RA cells of Aged atria, presumably underlying its effects on the plateau level of action potentials of the aged right atrial cells .
4.2 K+ currents of Aged atrial myocardium
Unlike Ca2+ currents, K+ currents appear to be augmented in Aged RA cells. This is in contrast to one report which described a decrease in Ito in aged rat ventricle , while another study of rat cells described an increase in Ito. Neither study controlled for the region of ventricle studied. We report here that the composite as well as TEA insensitive Ito increases with age in canine RA cells, and in composite current recordings (without TEA) Ito decay is slowed. Presumably this is secondary to the increased TEA sensitive Isus we report in these RA cells (Fig. 5D). Note that Isus is small in our Adult RA cells. This is dissimilar to findings of Yue et al.  and Feng et al.  where a prominent current, Ikur,d has been defined in atrial cells from dogs of unspecified ages. On the other hand, we define here a prominent Isus in RA cells from Aged dogs. These sustained currents inactivate with voltage (not shown), show rectification (Fig. 5) and are 4AP (50 μM) (not shown) and TEA sensitive (Fig. 6). Finally, TEA sensitive currents are significantly larger in the RA cells from Aged dogs and thus appear to result from the ‘normal’ aging process. Outward currents maintained at the end of reasonably short clamp steps have been observed in cells dispersed from human atria [9,27]. The molecular nature of the augmented Isus in Aged cells was not the focus of this study. However, based on its TEA sensitivity and the time course of these currents, it may be that currents through Kv3 potassium channel proteins contribute to both the transient and sustained atrial current. Kv3.1 protein has been shown to be present in dog atrial tissues . Clearly agents specifically acting on these Kv3.1 proteins would be expected to convert the Aged RA cell AP to one more similar to the Adult cell AP.
Our findings of both altered Ca2+ and K+ currents are consistent with the reported effects of the Ca2+ channel agonist BAY K8644 on action potentials of RA tissues . Importantly we show here that two major components of repolarization in cells of aged right atrial tissues are enhanced. Based on a computer model of an atrial cell , Ito and Isus augmentation should reduce APD30 consistent with the AP findings . However, augmented Isus cannot fully account for the altered APD90 and ERP observed in aged atrial tissues. Finally our findings are limited to studies of cells from one region of the RA and cannot necessarily be extended to cells of other regions of the aged atria.
Another limitation of the current study is that important cellular/ionic components of atrial conduction (gap junctional coupling, sodium channel function) were not studied. Further the extent to which the altered electrophysiologic properties seen with aging may be arrhythmogenic and increase the likelihood of Atrial Fibrillation (AF) is presently unknown. Certainly the depressed plateau and more gradual repolarization observed during phase 3 of the action potential would alter premature impulse propagation in Aged atria. Nevertheless, we conclude from these data that no matter what role these current changes play in the mechanism of AF, the pharmacology of the aged RA cell differs substantially from that of the adult RA cell.