QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.110080v1 319/2/898    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ehrlich, J. R. Articles by Gögelein, H. Search for Related Content PubMed PubMed Citation Articles by Ehrlich, J. R. Articles by Gögelein, H.

CARDIOVASCULAR

Properties of a Time-Dependent Potassium Current in Pig Atrium: Evidence for a Role of K_v1.5 in Repolarization

Division of Cardiology, J.W. Goethe-University, Frankfurt, Germany (J.R.E., C.H., S.H.H.); Research Center and Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada (P.C., S.N.); Genomic Sciences, Sanofi-Aventis, Frankfurt, Germany (C.M.-W., W.D.); Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada (S.N.); and Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (H.G.)

Received June 28, 2006; accepted August 15, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Cardiac electrical activity is modulated by potassium currents. Pigs have been used for antiarrhythmic drug testing, but only sparse data exist regarding porcine atrial ionic electrophysiology. Here, we used electrophysiological, molecular, and pharmacological tools to characterize a prominent porcine outward K⁺ current (I_K,PO) in atrial cardiomyocytes isolated from adult pigs. I_K,PO activated rapidly (time to peak at +60 mV; 2.1 ± 0.2 ms), inactivated slowly (_f = 45 ± 10; _s = 215 ± 28 ms), and showed very slow recovery (_f = 1.54 ± 0.73 s; _s = 7.91 ± 1.78 s; n = 9; 36°C). Activation and inactivation were voltage-dependent, and current properties were consistent with predominant K⁺ conductance. Neurotoxins (heteropodatoxin, hongatoxin, and blood depressing substance) that block K_v4.x, K_v1.1, -1.2, -1.3, and -3.4 in a highly selective manner as well as H₂O₂ and tetraethylammonium, did not affect the current. Drugs with K_v1.5-blocking properties (flecainide, perhexiline, and the novel atrial-selective antiarrhythmic 2'-{2-(4-methoxyphenyl)-acetylamino-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide; AVE0118) inhibited I_K,PO (IC₅₀ of 132 ± 47, 17 ± 10, and 1.25 ± 0.62 µM, respectively). 4-Aminopyridine suppressed the current and accelerated its decay, reducing charge carriage with an IC₅₀ of 39 ± 15 µM. Porcine-specific K_v channel subunit sequences were cloned to permit real-time quantitative reverse transcription-polymerase chain reaction on RNA extracted from isolated cardiomyocytes, which showed much greater abundance of K_v1.5 mRNA compared with K_v1.4, K_v4.2, and K_v4.3. Action potential recordings showed that I_K,PO inhibition with 0.1 mM 4-AP delayed repolarization (e.g., action potential duration at –50 mV increased from 45 ± 9 to 69 ± 5 ms at 3 Hz; P < 0.05). In conclusion, porcine atrium displays a current that is involved in repolarization, inactivates more slowly than classic transient outward current, is associated with strong K_v1.5 expression, and shows a pharmacological profile typical of K_v1.5-dependent currents.

Potassium currents [in particular transient outward currents (I_to) and I_Kur] are among the targets of novel atrialselective drugs developed for treating AF (Knobloch et al., 2004). Kinetic differences of activation, inactivation and recovery from inactivation discriminate between various potassium currents and are related to properties of the underlying ion channel subunits. The properties of I_to have been characterized extensively in many species (Patel and Campbell, 2005). Fast-inactivating I_to (I_to,f) is carried by K_v4.3 subunits in dog and human, whereas K_v1.4 subunits contribute to I_to,f in rabbit atria (Wang et al., 1999; Patel and Campbell, 2005). Rabbit I_to is characterized by particularly slow recovery from inactivation, which is related to the participation of K_v1.4 subunits (Wang et al., 1999). In human atrium, I_Kur is carried by K_v1.5 subunits, a member of the delayed rectifier current family, and has been shown to inactivate slowly over a period of seconds (Feng et al., 1998; Nattel et al., 1999). At room temperature, I_Kur may show a noninactivating phenotype (Li et al., 2004) although recording conditions importantly modulate inactivation kinetics of this current (Snyders et al., 1993). Likewise, -subunits may modify inactivation properties of K_v1.5 currents (Uebele et al., 1996).

Porcine atrial cellular electrophysiology has been studied to a limited extent and is poorly understood, although this species is commonly used in experimental studies (Janse et al., 1998). A recent investigation demonstrated the presence of a Ca²⁺-dependent chloride current (I_Cl,Ca) and I_Kur in porcine atria (Li et al., 2004). In preliminary studies, we noted a robust time-dependent current in porcine atrium that activates rapidly (like I_Kur) and inactivates more slowly than classic I_to and somewhat more rapidly than previously reported I_Kur. This study aimed to characterize this porcine outward current (which we abbreviate I_K,PO) with respect to electrophysiological properties, expression of potential corresponding underlying subunit transcripts, pharmacological responses, and functional role. In particular, we were interested in studying the pharmacological profile of the current with respect to known selective blockers of potential underlying K⁺ channel subunits, with a view to determining whether I_K,PO can potentially account for previous reports of anti-AF actions of K_v1.5 blockers in pig hearts.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animal and Tissue Handling. Male castrated pigs of the German landrace (n = 62; 19 ± 0.5 kg) were anesthetized with intravenous application of 30 mg/kg pentobarbital. During deep anesthesia, hearts were excised via left thoracotomy, resulting in humane euthanasia. Hearts were immediately immersed in oxygenated Tyrode's solution. All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication 85-13, revised 1996) and were performed by technicians specifically trained and experienced in animal care.

For isolation of single cardiomyocytes, the proximal circumflex coronary artery was cannulated and the atrial preparation was perfused with oxygenated Tyrode's solution on a Langendorff apparatus. The perfusion solution was then switched to Ca²⁺-free Tyrode's solution until all contraction ceased (10 min), and 100 U/ml collagenase (type II; Worthington Biochemicals, Freehold, NJ)-containing Ca²⁺-free Tyrode's solution was used for cell isolation as reported previously (Gogelein et al., 2004). After isolation, cells were stored in a high-[K⁺] storage solution at room temperature and studied within 12 h. Only healthy-looking cells with clear cross-striations and sharp edges were used for electrophysiological measurements. For real-time RT-PCR measurements, aliquots of isolated atrial cardiomyocytes were used, whereas the remainder of the cell isolation was used for electrophysiological experiments on the same days.

Solutions and Drugs. The high-[K⁺]-containing cell storage solution contained 120 mM KCl, 10 mM KH₂PO₄, 10 mM dextrose, 40 mM mannitol, 70 mM L-glutamic acid, 10 mM -OH-butyric acid, 20 mM taurine, 10 mM EGTA, and 0.1% bovine serum albumin (pH 7.3; KOH). Tyrode (extracellular) solution contained 136 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 0.33 mM NaH₂PO₄, 5 mM HEPES, and 10 mM dextrose, (pH 7.35 at 36°C; NaOH). CdCl₂ (200 µM; Sigma-Aldrich, St. Louis, MO) and 1 µM HMR 1556 (Sanofi-Aventis, Frankfurt, Germany; Gerlach et al., 2001) were added to suppress L-type calcium current and I_Ks. I_Na contamination was avoided by using a 50-ms prepulse to –50 mV or by substitution of equimolar Tris-HCl for external NaCl. The internal solution for current recording contained 110 mM K⁺-aspartate, 20 mM KCl, 1 mM MgCl₂, 5 mM MgATP, 0.1 mM Li-GTP, 10 mM HEPES, 5 mM Na-phosphocreatine, and 5 mM EGTA (pH 7.3; KOH). For action potential (AP) recording, EGTA was omitted. Fresh solutions were prepared daily. Drugs were from Sigma-Aldrich unless otherwise indicated, and toxins were from Alomone Labs (Jerusalem, Israel). Stock solutions were initially prepared and used throughout the study; for individual experiments, cells were incubated until steady-state current inhibition or washout was reached. Toxin-containing solutions were prepared fresh on each day of experimentation.

Data Acquisition and Analysis. Currents were recorded in voltage-clamp mode with whole-cell patch-clamp at 36 ± 0.5°C (Gogelein et al., 2004). Data sampling was performed at 1 kHz, and filtering was at 250 Hz. Borosilicate glass electrodes had tip resistances between 1.5 and 3.0 M when filled with internal solution. Mean ± S.E.M. compensated series-resistance was 5.6 ± 0.1 M. Cell capacitance averaged 47.6 ± 1.9 pF (n = 141). To control for cell size variability, currents were expressed as densities (pA/pF). Junction potentials between bath and pipette solution averaged 3.6 ± 0.5mV. APs were recorded in current-clamp mode and elicited with 2-ms twice threshold depolarizations.

Nonlinear algorithms were used for curve-fitting. t tests were used for two-group statistical comparisons. P < 0.05 indicated statistical significance. Data are expressed as mean ± S.E.M.

Cloning of Porcine K_v Channel Subunits. To define primers for real-time RT-PCR, partial DNA sequences for pig K_v4.3 (KCND3), K_v4.2 (KCND2), K_v1.4 (KCNA4), and KChIP2 (KCNIP2) were identified. The complete sequence for porcine K_v1.5 (KCNA5) has been reported (NM001006593) (Gogelein et al., 2004). PCR was first performed on pig brain (for KCND3, KCND2, and KCNA4) or heart (for KCNIP2) cDNA with degenerate primers based on sequences from other species. Pig-specific sequences were then used to design primers (Table 1) to amplify 660 bp (KCND3), 532 bp (KCND2), 630 bp (KCNA4), and 591 bp (KCNIP2) cDNA fragments. The fragments were cloned into pCRIIblunt or pCR2.1Topo vectors and sequenced [GenBank accession nos. DQ285632 [GenBank] (KCND3), DQ285631 [GenBank] (KCND2), DQ285633 [GenBank] (KCNA4), and DQ285634 [GenBank] (KCNIP2)].

Quantitative Real-Time RT-PCR. Isolated cardiomyocytes were homogenized (Mixermill 300; QIAGEN, Valencia, CA), and total RNA was extracted (QIAGEN). DNase digestion was performed with 10 µg of RNA, 5 µl of 10x DNase buffer (Ambion, Austin, TX), 1 µl of RNase inhibitor (Applera, Norwalk, CT), and 1 µl of DNaseI (2 U µl^–1; Ambion) in 50 µl. cDNA synthesis was performed from 2 µg of RNA (reverse transcriptase kit; Applera). Samples were incubated at 25°C for 10 min and 42°C for 60 min. The reaction was stopped by heating to 95°C for 5 min.

The RT product was then used as a template for subsequent PCR with gene-specific primers (Table 1). Real-time PCR was performed using an ABI Prism 7900 (Applera) and the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles at 95°C for 15 s, and 1 min at 60°C. Multiplex PCR used 0.125 µl of target probe (50 µM), 0.45 µl of target forward primer (50 µM), 0.45 µl of target reverse primer (50 µM), 12.5 µl of TaqMan 2x PCR master mix (Applera), 1.25 µl of 20x target primers and probes (PreDeveloped TaqMan assay reagents, 18S rRNA control; Applera), and 10 µl of cDNA sample (1:20 diluted with water) or used undiluted. RNA abundance was expressed as Ct, K_v subunit expression normalized to that of the internal control (18S) (Bustin, 2005).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Voltage and Time Dependence. One-second depolarizing pulses from a holding potential of –80 mV to potentials between –60 and +60 mV (0.1 Hz; Fig. 1A) elicited rapidly activating outward currents showing time-dependent inactivation. Based on this observation and further evidence detailed below, we termed this current I_K,PO for porcine outward potassium current. I_K,PO amplitude was quantified as the difference between peak and end-pulse steady-state current unless stated otherwise. Threshold to current activation was positive to –20 mV, and myocytes had a mean ± S.E.M. I_K,PO-density of 11.6 ± 1.6 pA/pF upon depolarization to +60 mV (n = 20; Fig. 1B).

View larger version (33K): [in this window] [in a new window]   Fig. 1. Biophysical current characterization. A, IK,PO recorded with 1000-ms depolarizations from a holding potential of –80 mV to potentials between 0 and +60 mV (protocol in inset, 0.1 Hz). B, mean ± S.E.M. IK,PO-voltage relationship (n = 20). C, voltage dependence of steady-state inactivation with 1000-ms prepulses to various potentials and 750-ms test pulses to +60 mV. D, mean ± S.E.M. data for voltage dependence of IK,PO activation and inactivation (n = 10 each). E, mean ± S.E.M. time to peak IK,PO and inactivation time constants (n = 10 each). F, example of IK,PO recovery from inactivation. G, mean ± S.E.M. current during the second pulse (IP2) normalized to current during the first pulse (IP1), as a function of P1-P2 interval (protocol in inset) with biexponential fit to mean ± S.E.M. data (n = 9). H, IK,PO during the 10th pulse (IP10) to +60 mV normalized to that during first pulse (IP1) plotted over different frequencies with and without extracellular sodium. Act., activation; inact., inactivation; f, fast time constant; s, slow time constant; TP, test potential.

Current activation-speed assessed as time to peak was voltage dependent and became faster at more positive potentials (e.g., 13.5 ± 1.8 ms at 0 mV, 2.1 ± 0.24 ms at +60 mV; Fig. 1E). Inactivation kinetics were best fitted by biexponential functions, and resulting time constants () were slow (e.g., at +60 mV, _f averaged 45 ± 10 and _s was 215 ± 28 ms; n = 10; Fig. 1E).

Recovery from inactivation was assessed with a paired pulse protocol with depolarizations (P1 and P2) to +60 mV at increasing P1-P2 intervals (Fig. 1F) and a holding potential of –80 mV. Current during P2 was normalized to current during P1 and showed biexponential recovery with time constants of 1.54 ± 0.73 s (_f) and 7.91 ± 1.78 s (_s; n = 9; Fig. 1G).

To study frequency dependence, cells were repetitively depolarized from –80 to +60 mV (410-ms pulses) in Tris-Cl-containing, Na⁺-free external solution. Currents during the 10th pulse were normalized to current during the first pulse, showing a frequency-dependent decline (n = 15; Fig. 1H). Similar experiments were performed in the presence of extracellular NaCl with 50-ms prepulses to –50 mV to inactivate I_Na. These recordings showed slightly greater frequency dependence, but they were qualitatively comparable with those obtained with Tris-Cl (n = 7; Fig. 1H). In Tris-Cl, I_K,PO elicited with the 10th pulse were 46 ± 3% (0.5 Hz) of P1, declining to 13 ± 3% (2 Hz), compared with 38 ± 3% (0.5 Hz) and 7 ± 1% (2 Hz) in NaCl-containing solution (P = 0.07 and 0.05, respectively). The effect of equimolar Na⁺ replacement by Tris and prepulse protocols to inactivate I_Na in Na⁺ - containing solution on peak to steady-state current was subtle. In nine cells, I_K,PO at +60 mV without prepulses averaged 8.3 ± 1.4 and 9.5 ± 1.4 pA/pF (P = N.S.) in the presence and absence of extracellular Na⁺, respectively. In Na⁺-containing Tyrode's solution, currents recorded at +60 mV with 50-ms prepulses to –50 mV, averaged 8.4 ± 1.5 compared with 9.3 ± 1.4 pA/pF without prepulses (P = N.S.).

Effects of Cl^– and K⁺ Substitution on I_K,PO. To assess the potential charge carrier of I_K,PO, we investigated the effects of Cl^– and K⁺ substitution on the current under study. The reversal potential of the current averaged –69.2 ± 2.3 mV (n = 3 cells). After correction for liquid-junction potentials this value was 72 mV, compatible with a predominantly K⁺-carried conductance.

I_K,PO was also recorded in individual cells before and after replacement of external chloride by equimolar cyclamate to assess potential contributions of a previously described Ca²⁺-dependent Cl^– current (Li et al., 2004). This intervention did not alter I_K,PO (Fig. 2, A and B). Mean ± S.E.M. I_K,PO density was similar over a range of voltages, averaging 7.1 ± 1.8 pA/pF at +60 mV in NaCl and 7.0 ± 1.9 pA/pF with Nacyclamate (n = 5; P = N.S.; Fig. 2C). The effects of intracellular K⁺ replacement by Cs⁺ were then determined. Currents were recorded from 10 cells with regular internal solution and from nine other cells isolated from the same pigs on the same days with equimolar substitution of CsCl for internal KCl. Intracellular K⁺ removal abolished I_K,PO: currents at +60 mV averaged 12.1 ± 0.9 pA/pF (KCl) versus 0.2 ± 0.05 pA/pF (CsCl; P < 0.001; Fig. 2F). Together, these results strongly suggest that I_K,PO is primarily a K⁺ current.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Determination of Cl– and K+ dependence. A and B, IK,PO recorded from the same cardiomyocyte before (A) and after (B) substitution of external chloride with equimolar cyclamate (protocol in inset). C, mean ± S.E.M. data (n = 5). D, IK,PO-recording obtained at +60 mV with regular K+-containing internal solution. E, IK,PO-recording with substitution of internal K+ with equimolar Cs+ from a different cell isolated from the same pig as in D on the same day. F, mean ± S.E.M. IK,PO density at +60 mV for KCl (n = 10) and for CsCl (n = 9) (P < 0.001). TP, test potential.

Pharmacological Characterization. After having characterized the current as a K⁺-dependent current with slow inactivation and recovery from inactivation, we set out to obtain the pharmacological profile of I_K,PO. The K⁺ channel blocker 4-aminopyridine (4-AP) was applied at concentrations between 0.1 µM and 100 mM. The left panel of Fig. 3A illustrates 4-AP effects on I_K,PO. Reversible suppression was seen, with an IC₅₀ on peak to steady-state current of 0.81 ± 0.16 mM (n = 7; Fig. 3A, right, closed circles). Washout returned current amplitude to 81 ± 5% of control. 4-AP at lower concentrations significantly accelerated current inactivation: inactivation _s and _f averaged was 135 ± 70 and 35 ± 14 ms, respectively, after application of 100 µM 4-AP, compared with 315 ± 51 and 115 ± 7 ms, respectively, under control conditions (P < 0.05 for each), a behavior suggestive of open channel block (Fedida, 1997). To consider the reduction of charge carried by I_K,PO, we determined the 4-AP IC₅₀ based on the area under the current-time curve (Dukes et al., 1990; Gogelein et al., 2004). Integration of the area between the remaining current at maximal 4-AP concentration and the transient outward currents for determination of fractional block yielded an IC₅₀ of 39 ± 15 µM (Fig. 3A, closed circles; P < 0.01).

View larger version (34K): [in this window] [in a new window]   Fig. 3. Pharmacological properties of IK,PO. A, left, IK,PO recorded with depolarizations to +60 mV (protocol in inset) under control conditions and after the application of ascending extracellular 4-AP concentrations (only recordings at 100 µM to 10 mM shown). Note the acceleration of current inactivation with low concentrations of 4-AP suggestive of open channel block. Right, mean ± S.E.M. fractional block for peak to steady-state current (open symbols) and integrated area under the curve (AUC; closed symbols). B, left, example of IK,PO inhibition by flecainide (traces shown are from 10 µM to 1 mM); right, mean ± S.E.M. fractional block. C, representative currents recorded before and after the application of 0.01% H2O2 (protocol in inset). Right, mean ± S.E.M. IK,PO-voltage relations (n = 3). D, left, IK,PO recordings before and after the application of 10 mM TEA and mean ± S.E.M. data (n = 9; right). Fits shown are to mean ± S.E.M. data. All protocols were delivered at 0.1 Hz. Dashed lines represent zero-current level; CTL, control; TP, test potential.

We next determined the effect of flecainide (a moderately potent blocker of K_v1 channels) on I_K,PO. Flecainide was applied at 100 nM to 1 mM (Fig. 3B, left) and suppressed I_K,PO with an IC₅₀ of 132 ± 47 µM (n = 10; Fig. 3B, right). The effect was reversible upon washout (83 ± 12% of control). The inactivation of K_v1.4 is substantially slowed by oxidative stress as imposed by H₂O₂ (Dixon et al., 1996). We applied H₂O₂ at an external concentration of 0.01% (Fig. 3C) and found no effect of H₂O₂ on I_K,PO inactivation (e.g., at +60 mV, _s = 292 ± 19 ms before versus 299 ± 15 ms after H₂O₂; P = N.S.). Likewise, current amplitude remained unaffected and averaged 8.4 ± 4.5 pA/pF (at +60 mV) before versus 8.2 ± 4.5 pA/pF after H₂O₂ arguing against a significant role for K_v1.4. Tetraethylammonium (TEA; 10 mM) did not significantly affect peak to steady-state I_K,PO, e.g., under control conditions, current density at +60 mV averaged 13.6 ± 1.6 compared with 15.0 ± 1.7 pA/pF with TEA and 14.9 ± 2.1 pA/pF after 15 min washout (Fig. 3D; n = 9; P = N.S.).

Effects of Specific Neurotoxins. The slow inactivation of I_K,PO made an important contribution of K_v4 subunits unlikely. To assess further any possible K_v4 contribution, heteropodatoxin (a potent blocker of native I_to,1 and heterologously expressed K_v4 channels; Sanguinetti et al., 1997) was applied at 100 and 500 nM. Heteropodatoxin had no effect on I_K,PO [e.g., at +60 mV, mean ± S.E.M. control current density was 14.2 ± 5.3 versus 16.2 ± 6.7 (100 nM) and 15.9 ± 6.6 pA/pF (500 nM) (n = 7; P = N.S.)]. Hongatoxin blocks heterologously expressed K_v1.1, -1.2, and -1.3 channels with IC₅₀ values of 31, 170, and 86 pM, respectively (Koschak et al., 1998). No change in I_K,PO was seen with 0.1 nM (e.g., at +60 mV, mean ± S.E.M. I_K,PO was 32.9 ± 5.6 before versus 32.5 ± 5.8 pA/pF after hongatoxin, respectively; n = 5; P = N.S.). Blood depressing substance is an Anemonia sulcata toxin that blocks K_v3.4 transient outward currents (Diochot et al., 1998). I_K,PO density at +60 mV in six cells averaged 26.4 ± 4.8 before versus 26.7 ± 5.0 pA/pF after application of 100 nM blood depressing substance (P = N.S.).

After the pharmacological exclusion of a significant contribution of K_v4, K_v1.1, -1.2, -1.3, and -3.4 subunits, we assessed the response to K_v1.5 blockers. We first studied the effect of perhexiline (an antianginal drug that inhibits heterologously expressed K_v1.5 with an IC₅₀ of 1.5 µM) (Rampe et al., 1995). Figure 4A depicts representative currents recorded under control conditions and in the presence of perhexiline. I_K,PO was clearly suppressed (IC₅₀ of 17 ± 10 µM; n = 9; Fig. 4B). The atrial-selective compound AVE0118 suppresses K_v1.5 current in heterologous systems with an IC₅₀ of 1.1 ± 0.2 µM (Gogelein et al., 2004). AVE0118 inhibited I_K,PO with an IC₅₀ of 1.25 ± 0.62 µM (Fig. 4, C and D). Consistent with open channel block, _f. accelerated from 76 ± 18 ms (control) to 17 ± 2 ms with 1 µM AVE0118 (n = 8; P < 0.05), whereas _s remained unaltered (_s = 332 ± 26 versus 327 ± 53 ms for control and AVE0118, respectively; P = N.S.).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Inhibition by Kv1.5 blockers. Effects of ascending perhexiline concentrations on IK,PO are shown in A (protocol in inset) with mean ± S.E.M. fractional block (n = 9; B). C, representative currents from a cell exposed to various concentrations of AVE0118. D, mean ± S.E.M. fractional block (n = 8). Fits shown are to mean ± S.E.M. data.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Consequence of IK,PO blockade on action potentials. Effect of externally applied 0.1 mM 4-AP on APs recorded from single cells in current-clamp mode. A, representative APs before and after 4-AP application. Washout in dashed lines. B, mean ± S.E.M. AP duration measured at –50 and –70 mV (n = 5). *, P < 0.05.

View larger version (32K): [in this window] [in a new window]   Fig. 6. mRNA expression of Kv channel subunits. Relative expression of Kv subunits normalized to the expression of 18S. A, mean ± S.E.M. data from six animals. B, representative amplification plots of real-time PCR reactions (double reaction sets) for the subunits tested. The indicator line was individually adjusted to obtain the respective Ct value for each experiment. *, P < 0.001.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Previous Studies on Porcine Electrophysiology. Pigs have been used for a variety of experimental studies of cardiac arrhythmias (Janse et al., 1998; Wirth et al., 2003), but information about the cardiac cellular electrophysiology of the pig is limited. Porcine sinoatrial cells exhibit I_Ks (Ono et al., 2000) and ventricular myocytes exhibit an I_Cl,Ca that contributes to repolarization (Li et al., 2003). Another study by the latter investigators documented the presence of I_Cl,Ca, I_Kur, I_Kr, and I_Ks in pig atrial myocytes (Li et al., 2004). The I_Kur (which the authors called I_Kur.p) reported in the latter study was relatively small and apparent primarily at slow frequencies (0.05 Hz) at room temperature. I_Kur.p showed weak inward rectification, use dependence, 4-AP sensitivity (IC₅₀ of 72 ± 4 µM), and TEA resistance. This work differs from ours in the use of low EGTA concentration in the pipette and recording at room temperature.

Our study adds to previous results in showing the presence of a substantial time-dependent outward K⁺ current that is sensitive to blockers of K_v1.5, but not other possible underlying subunits, and that contributes to porcine atrial repolarization. Several lines of additional evidence presented here (including biophysical properties as well as mRNA expression) are consistent with a potential role for underlying K_v1.5 K⁺ channel subunits.

Relation of Biophysical Properties to Other Transient Outward Currents. The major candidate K⁺ channel subunits generating rapidly activating and inactivating (so called "fast") I_to phenotypes are K_v4.2 and K_v4.3 (Nerbonne, 2000; Oudit et al., 2001). Thus, the predominant subunit underlying native I_to in human ventricular cells is K_v4.3, which generates a current that inactivates rapidly as a single exponential process (_inact. = 7.9 ± 0.3 ms; 35°C) (Nabauer et al., 1996). Canine I_to,f is similarly carried by K_v4.3 subunits (Dixon et al., 1996). Rat ventricular I_to (predominantly K_v4.2) also inactivates rapidly (_inact. = 48 ± 7 ms; 22°C) (Himmel et al., 1999). In contrast, a functionally distinct I_to phenotype (I_to,slow) has been identified in many mammalian species, which inactivates with a double exponential process (_slow in the order of hundreds of milliseconds) and is carried by K_v1.4 subunits (Patel and Campbell, 2005). This I_to phenotype is distinguished from K_v4-based currents by its slow recovery from inactivation, with time constants on the order of seconds (Xu et al., 1999; Wickenden et al., 1999). For example, ferret I_to,slow recovers with = 3.0 ± 0.45 s (at 22°C) and mouse I_to,slow recovers with similar time constants (Brahmajothi et al., 1999; Xu et al., 1999).

Relation of Biophysical Properties to I_Kur and I_K,slow. Mouse ventricular myocytes express a current termed I_K,slow with kinetic properties consistent with K_v1.5 -subunits (Zhou et al., 1998). In other species, K_v1.5 underlies the atrially expressed ultrarapid delayed rectifier current (I_Kur), which is often described as noninactivating. Although K_v1.5 current has generally been described as a delayed rectifier, it can show substantial time-dependent inactivation, with complete inactivation for depolarizations of sufficient duration (Feng et al., 1998; Lin et al., 2001; Snyders et al., 1993). The inactivation kinetics that we found for I_K,PO were faster than those published for hK_v1.5 in heterologous systems (e.g., _f = 250 ms; _s = 1500 ms; Lin et al., 2001), although the latter studies were performed at room temperature, which in itself substantially slows inactivation (Snyders et al., 1993). It is also possible that I_K,PO involves a contribution of -subunits, which are known to interact with K_v1.5 and accelerate its inactivation (Uebele et al., 1998). A full study of the molecular biology of I_K,PO would be very interesting, but it is beyond the scope of the present article.

Pharmacological Profile of I_K,PO. K_v1.5-based currents are sensitive to 4-AP. For example, mouse ventricular I_K,slow is inhibited by 4-AP with an IC₅₀ of 32 ± 5 µM (Zhou et al., 1998). Significant interspecies differences in 4-AP sensitivity of I_to currents attributed to K_v1.5 exist, with IC₅₀ values ranging up to 600 µM in rat atrium (Zhou et al., 1998). However, despite differences in affinity, all K_v1.5-carried currents are 4-AP sensitive, as was I_K,PO in this study. Relevant I_K,PO charge carriage inhibition (based on assessment of area under the current-time curve accounting for open channel block) occurred with an IC₅₀ of 39 ± 15 µM. IC₅₀ for heterologously expressed hK_v1.5 peak currents ranges between 50 and 290 µM (Grissmer et al., 1994; Bouchard and Fedida, 1995), consistent with the results for I_K,PO in the present study. Although 4-AP is nonspecific in that it blocks both I_to,f and I_to,s, the underlying mechanisms are distinct. Block of I_to,s occurs predominantly in the open state (Campbell et al., 1993). In contrast, 4-AP block of I_to,f occurs through closed state binding and displays use-dependent unblocking and reverse use dependence (Patel and Campbell, 2005). No use-dependent unblocking was observed in the present study, and block was consistent with open-state dependence. Both K_v1.4 and K_v1.5 currents show predominant open-state 4-AP block, but K_v1.4 is insensitive to flecanide (Akar et al., 2004) and sensitive to H₂O₂, inconsistent with the response of I_K,PO. A contribution of other K_v1 subunits to I_K,PO was excluded by the absence of any effect of hongatoxin application. In contrast, I_K,PO was sensitive to perhexiline, AVE0118, flecainide, and 4-AP at concentrations fully compatible with K_v1.5 inhibition (Rampe et al., 1995; Zhou et al., 1998; Gogelein et al., 2004). A relevant contribution of K_v3.1 subunits that have been shown to underlie I_Kur in dogs is excluded, because this current is exquisitely sensitive to TEA (IC₅₀ of 0.3 mM).

Potential Importance of Our Findings. Our findings provide detailed information about the pharmacological and biophysical characteristics of a porcine outward potassium current that is a candidate to mediate the reported effects of K_v1.5-inhibiting atrial antiarrhythmic drugs (Wirth et al., 2003).

Class III antiarrhythmic agents that delay atrial repolarization are effective in treating AF. However, previously developed class III antiarrhythmic agents have prolonged atrial repolarization by blocking I_Kr. The untoward side effects of I_Kr-inhibiting drugs include potentially lethal ventricular proarrhythmia (Hohnloser and Singh, 1995). The differential expression pattern of cardiac ion channel subunits (like K_v1.5) in atria versus ventricles provides a potential basis for treatment options for atrial arrhythmias that have reduced proarrhythmic risks (Nattel et al., 1999). The development of K_v1.5-based drugs as atrial antiarrhythmic agents has been limited by a lack of animal models with K_v1.5-regulated atrial repolarization. Porcine models have been used for in vivo testing of K_v1.5-blocking drugs and have demonstrated potent efficacy against atrial arrhythmias without significant ventricular actions (Wirth et al., 2003). I_K,PO, as characterized in this study, contributes to atrial repolarization and has properties suggesting that it is carried by K_v1.5 -subunits. This result provides for the first time a biophysical basis supporting the use of pigs as a model to test novel K_v1.5-inhibiting atrial-selective anti-AF agents.

Limitations of This Study. Variability in cell isolation can affect the results obtained. To minimize errors introduced by this process, we studied APs and mRNA levels from cells in pigs that were used for current recordings on the same day. Furthermore, native cells express a variety of ionic currents, and their electrophysiological isolation requires selective protocols and pharmacological agents with imperfect specificity. One-second pulses were chosen to allow for almost complete inactivation of the current. In some instances, incomplete inactivation might have caused biophysical inaccuracy, but longer pulses were poorly tolerated, and the results had minimal effect on the analyses. Another limitation of this study is the lack of protein expression data. We tried to obtain Western blots from porcine atrial protein preparations, but we were unable to obtain specific bands with commercially available antibodies, none of which have been raised against porcine-specific epitopes.

	Footnotes

 
J.R.E. was supported by Nachlass Martha Schmelz, Dr. Paul and Cilli Weill-Stiftung, and Deutsche Forschungsgemeinschaft (EH201/2-1). The contributions of P.C. and S.N. were supported by grants from Natural Sciences and Engineering Research Council, Canadian Institutes of Health Research, and the Quebec Heart and Stroke Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.110080.

ABBREVIATIONS: AF, atrial fibrillation; I_to, transient outward current; I_Kur, ultrarapid delayed rectifier K⁺ current; I_to,f, fast-inactivating transient outward current; I_Ks, slow delayed rectifier K⁺-potassium current; I_Na, voltage-gated sodium current; I_K,PO, porcine outward potassium current; AP, action potential; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); 4-AP, 4-aminopyridine; TEA, tetraethylammonium; AVE0118, 2'-{2-(4-methoxy-phenyl)-acetylamino-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide; HMR 1556, (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methylmethanesulfonamide; pA/pF, current amplitude normalized to cell capacitance; I_to, slow-inactivating transient outward current.

Address correspondence to: Dr. Joachim R. Ehrlich, Division of Cardiology, J.W. Goethe-University, Theodor Stern Kai 7, 60590 Frankfurt, Germany. E-mail: j.ehrlich{at}em.uni-frankfurt.de

	References

Top Abstract Materials and Methods Results Discussion References

 

Akar FG, Wu RC, Deschenes I, Armoundas AA, Piacentino V 3rd, Houser SR, and Tomaselli GF (2004) Phenotypic differences in transient outward K⁺ current of human and canine ventricular myocytes: insights into molecular composition of ventricular I_to. Am J Physiol 286: H602–H609.

Bouchard R and Fedida D (1995) Closed- and open-state binding of 4-aminopyridine to the cloned human potassium channel Kv1.5. J Pharmacol Exp Ther 275: 864–876.[Abstract/Free Full Text]

Brahmajothi MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne JM, and Strauss HC (1999) Distinct transient outward potassium current (I_to) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol 113: 581–600.[Abstract/Free Full Text]

Bustin SA (2005) Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences. Expert Rev Mol Diagn 5: 493–498.[CrossRef][Medline]

Campbell DL, Qu Y, Rasmusson RL, and Strauss HC (1993) The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine. J Gen Physiol 101: 603–626.[Abstract/Free Full Text]

Diochot S, Schweitz H, Beress L, and Lazdunski M (1998) Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4. J Biol Chem 273: 6744–6749.[Abstract/Free Full Text]

Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, and McKinnon D (1996) Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res 79: 659–668.[Abstract/Free Full Text]

Dukes ID, Cleemann L, and Morad M (1990) Tedisamil blocks the transient and delayed rectifier K⁺ currents in mammalian cardiac and glial cells. J Pharmacol Exp Ther 254: 560–569.[Abstract/Free Full Text]

Fedida D (1997) Gating charge and ionic currents associated with quinidine block of human Kv1.5 delayed rectifier channels. J Physiol (Lond) 499: 661–675.[CrossRef][Medline]

Feng J, Xu D, Wang Z, and Nattel S (1998) Ultrarapid delayed rectifier current inactivation in human atrial myocytes: properties and consequences. Am J Physiol 275: H1717–H1725.

Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Bruggemann A, Busch AE, Suessbrich H, Bleich M, and Greger R (2001) Synthesis and activity of novel and selective I(Ks)-channel blockers. J Med Chem 44: 3831–3837.[CrossRef][Medline]

Gogelein H, Brendel J, Steinmeyer K, Strubing C, Picard N, Rampe D, Kopp K, Busch AE, and Bleich M (2004) Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels. Naunyn-Schmiedeberg's Arch Pharmacol 370: 183–192.[Medline]

Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin DD, and Chandy KG (1994) Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol 45: 1227–1234.[Abstract]

Himmel HM, Wettwer E, Li Q, and Ravens U (1999) Four different components contribute to outward current in rat ventricular myocytes. Am J Physiol 277: H107–H118.

Hohnloser SH and Singh BN (1995) Proarrhythmia with class III antiarrhythmic drugs: definition, electrophysiologic mechanisms, incidence, predisposing factors, and clinical implications. J Cardiovasc Electrophysiol 6: 920–936.[Medline]

Janse MJ, Opthof T, and Kleber AG (1998) Animal models of cardiac arrhythmias. Cardiovasc Res 39: 165–177.[Free Full Text]

Knobloch K, Brendel J, Rosenstein B, Bleich M, Busch AE, and Wirth KJ (2004) Atrial-selective antiarrhythmic actions of novel I_kur vs. I_kr, I_ks, and I_KAch class Ic drugs and beta blockers in pigs. Med Sci Monit 10: BR221–BR228.[Medline]

Koschak A, Bugianesi RM, Mitterdorfer J, Kaczorowski GJ, Garcia ML, and Knaus HG (1998) Subunit composition of brain voltage-gated potassium channels determined by hongotoxin-1, a novel peptide derived from Centruroides limbatus venom. J Biol Chem 273: 2639–2644.[Abstract/Free Full Text]

Li GR, Du XL, Siow YL, O K, Tse HF, and Lau CP (2003) Calcium-activated transient outward chloride current and phase 1 repolarization of swine ventricular action potential. Cardiovasc Res 58: 89–98.[Abstract/Free Full Text]

Li GR, Sun H, To J, Tse HF, and Lau CP (2004) Demonstration of calcium-activated transient outward chloride current and delayed rectifier potassium currents in Swine atrial myocytes. J Mol Cell Cardiol 36: 495–504.[CrossRef][Medline]

Lin S, Wang Z, and Fedida D (2001) Influence of permeating ions on Kv1.5 channel block by nifedipine. Am J Physiol 280: H1160 –H1172.

Nabauer M, Beuckelmann DJ, Uberfuhr P, and Steinbeck G (1996) Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation 93: 168–177.[Abstract/Free Full Text]

Nattel S and Opie LH (2006) Controversies in atrial fibrillation. Lancet 367: 262–272.[CrossRef][Medline]

Nattel S, Yue L, and Wang Z (1999) Cardiac ultrarapid delayed rectifiers: a novel potassium current family o f functional similarity and molecular diversity. Cell Physiol Biochem 9: 217–226.[CrossRef][Medline]

Nerbonne JM (2000) Molecular basis of functional voltage-gated K⁺ channel diversity in the mammalian myocardium. J Physiol (Lond) 525: 285–298.[Abstract/Free Full Text]

Ono K, Shibata S, and Iijima T (2000) Properties of the delayed rectifier potassium current in porcine sino-atrial node cells. J Physiol (Lond) 524: 51–62.[Abstract/Free Full Text]

Oudit GY, Kassiri Z, Sah R, Ramirez RJ, Zobel C, and Backx PH (2001) The molecular physiology of the cardiac transient outward potassium current (I(to)) in normal and diseased myocardium. J Mol Cell Cardiol 33: 851–872.[CrossRef][Medline]

Patel SP and Campbell DL (2005) Transient outward potassium current, `Ito', phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms. J Physiol (Lond) 569: 7–39.[Abstract/Free Full Text]

Rampe D, Wang Z, Fermini B, Wible B, Dage RC, and Nattel S (1995) Voltage- and time-dependent block by perhexiline of K⁺ currents in human atrium and in cells expressing a Kv1.5-type cloned channel. J Pharmacol Exp Ther 274: 444–449.[Abstract/Free Full Text]

Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, and Mueller AL (1997) Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol 51: 491–498.[Abstract/Free Full Text]

Snyders DJ, Tamkun MM, and Bennett PB (1993) A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression. J Gen Physiol 101: 513–543.[Abstract/Free Full Text]

Tseng GN, Jiang M, and Yao JA (1996) Reverse use dependence of Kv4.2 blockade by 4-aminopyridine. J Pharmacol Exp Ther 279: 865–876.[Abstract/Free Full Text]

Uebele VN, England SK, Chaudhary A, Tamkun MM, and Snyders DJ (1996) Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits. J Biol Chem 271: 2406–2412.[Abstract/Free Full Text]

Uebele VN, England SK, Gallagher DJ, Snyders DJ, Bennett PB, and Tamkun MM (1998) Distinct domains of the voltage-gated K⁺ channel Kv beta 1.3 beta-subunit affect voltage-dependent gating. Am J Physiol 274: C1485–C1495.

Wang Z, Feng J, Shi H, Pond A, Nerbonne JM, and Nattel S (1999) Potential molecular basis of different physiological properties of the transient outward K⁺ current in rabbit and human atrial myocytes. Circ Res 84: 551–561.[Abstract/Free Full Text]

Wickenden AD, Jegla TJ, Kaprielian R, and Backx PH (1999) Regional contributions of Kv1.4, Kv4.2, and Kv4.3 to transient outward K⁺ current in rat ventricle. Am J Physiol 276: H1599 –H1607.

Wirth KJ, Paehler T, Rosenstein B, Knobloch K, Maier T, Frenzel J, Brendel J, Busch AE, and Bleich M (2003) Atrial effects of the novel K(+)-channel-blocker AVE0118 in anesthetized pigs. Cardiovasc Res 60: 298–306.[Abstract/Free Full Text]

Wolf PA, Mitchell JB, Baker CS, Kannel WB, and D'Agostino RB (1998) Impact of atrial fibrillation on mortality, stroke, and medical costs. Arch Intern Med 158: 229–234.[Abstract/Free Full Text]

Xu H, Guo W, and Nerbonne JM (1999) Four kinetically distinct depolarizationactivated K⁺ currents in adult mouse ventricular myocytes. J Gen Physiol 113: 661–678.[Abstract/Free Full Text]

Zhou J, Jeron A, London B, Han X, and Koren G (1998) Characterization of a slowly inactivating outward current in adult mouse ventricular myocytes. Circ Res 83: 806–814.[Medline]

This article has been cited by other articles:

				J. R. Ehrlich, P. Biliczki, S. H. Hohnloser, and S. Nattel Atrial-Selective Approaches for the Treatment of Atrial Fibrillation J. Am. Coll. Cardiol., February 26, 2008; 51(8): 787 - 792. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.110080v1 319/2/898    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ehrlich, J. R. Articles by Gögelein, H. Search for Related Content PubMed PubMed Citation Articles by Ehrlich, J. R. Articles by Gögelein, H.