Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Delayed rectifier K currents have reduced amplitudes and altered kinetics in myocytes from infarcted canine ventricle

Delayed rectifier K currents have reduced amplitudes and altered kinetics in myocytes from... Abstract Objective: The rapid (IKr) and slow (IKs) components of delayed rectifier currents play an important role in determining the cardiac action potential configuration. Abnormalities in their function may contribute to arrhythmogenesis under pathological conditions. We studied the effects of myocardial infarction on IKr and IKs in canine ventricular myocytes and their molecular basis. Methods: Infarct zone myocytes (IZs) were isolated from a thin layer of surviving epicardium overlying an infarct 5 days after a total occlusion of the left anterior descending (LAD) coronary artery. Normal myocytes (NZs) were isolated from the corresponding region of control hearts for comparison. Currents were recorded under the whole-cell patch clamp conditions. Results: Both IKr and IKs current densities were reduced in IZs versus NZs. Kinetic analysis further suggests an acceleration of IKr activation and IKs deactivation. RNase protection assays were used to quantify the mRNA levels of IKr and IKs channel subunits (dERG, dIsK and dKvLQT1) in tissue immediately adjacent to the region where myocytes were isolated. mRNA levels of all three subunits were reduced 2 days after LAD occlusion (by 48±9%, 68±5%, and 45±4% for dERG, dIsK and dKvLQT1, respectively, n = 8 each). By day 5, the dKvLQT1 message returned to control while those of dERG and dIsK remained reduced (by 52±7% and 76±6%, respectively). Conclusions: The decrease in IKr and IKs amplitudes and changes in their kinetics in infarcted tissue might be due to a decrease in functional channels and/or changes in their subunit composition. Heterogeneous changes in IKr and IKs in infarcted hearts may impact on the effects of varying heart rate or neurohumoral modulation on repolarization. K-channel, Myocytes, Infarction, Gene expression, Remodeling, Single channel currents Time for primary review 23 days. This article is referred to in the Editorial by M.W. Veldkamp (pages 11–22) in this issue. 1 Introduction A canine model of myocardial infarction (MI) created by total occlusion of the left anterior descending (LAD) coronary artery has been extensively studied [1,2]. This model exhibits many features of arrhythmias similar to observations of patients with MI [3]. Thus it is a useful model for studying mechanisms of arrhythmias post MI. In this model, 4 to 5 days after LAD occlusion there is a layer of surviving epicardium of varying thickness overlying the infarct (epicardial border zone or EBZ) [1]. Ventricular tachyarrhythmias induced by programmed stimulation often result from reentrant circuits formed within the EBZ [3]. Although perturbations in cellular electrophysiology of the EBZ have been well characterized and linked to arrhythmogenesis, there is an important omission in the previous studies: the effects of MI on the delayed rectifier currents have not been examined. There are two components of delayed rectifier currents: the rapid (IKr) and the slow (IKs) components [4,5]. Both have been described for human atrial and ventricular myocytes [6,7] and may participate in action potential repolarization, yet relatively little is known about how disease processes affect their function and expression. In this study, we quantified and characterized IKr and IKs in myocytes isolated from the EBZ (IZ) and from the corresponding region of normal hearts (NZ). In addition, we quantified the mRNA levels of three putative subunits of IKr and IKs channels (dERG, dIsK, and dKvLQT1, ‘d’ for ‘dog’ isoforms) [8–10]. dERG and dKvLQT1 are the pore-forming subunits [8,10], while dIsK (also called minK) is a regulatory subunit that can associate with dKvLQT1 or dERG and affect their channel function [9,10]. Our data showed that there was a reduction in the mRNA levels of these subunits in infarcted hearts, but the degree of reduction was not the same among the three. In particular, we observed an uneven change in dKvLQT1 and dIsK mRNA levels that may lead to alterations in IKs channel behavior (gating kinetics and pharmacology) manifested at a later time (<5 days) after LAD occlusion. 2 Methods 2.1 Surgery Experiments on animals were conducted in conformity with the Declaration of Helsinki (Br Med J 1964; ii: 177) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The surgical procedure was as described [2]. Two or five days later, the infarct zone tissue (EBZ) was removed for myocyte isolation and RNA preparation. Tissue from the corresponding region of normal hearts was used as a control. 2.2 Electrophysiology 2.2.1 Myocyte preparation Ventricular myocytes were enzymatically dispersed as previously described [2]. For NZs, only rod-shaped cells with clear striations and surface free of blebs were used for voltage clamp. IZs were chosen for study based on the following morphological criteria: a ruffled appearance, not very clear striations, irregular shape, and small dark droplets [2]. 2.2.2 Voltage clamp experiments Whole-cell currents were recorded using the conventional suction pipette method at 24–26°C. About 6 min after forming the whole-cell recording (WCR) configuration, the bath solution was switched to a nominally Na- and Ca-free solution for 6 min, after which data collection began (see Fig. 1B). Fig. 1 Open in new tabDownload slide (A) Currents from an NZ and an IZ before (control) and after addition of azimilide. Inset: voltage clamp protocol, holding voltage (Vh) −50 mV, test voltages (Vt) −40 to +70 mV in 10 mV increments for 5 s once every 30 s. Doted lines: zero current level. Arrowheads: step increase in current amplitude upon depolarization to +70 mV. (B) Time course of changes in delayed rectifier current amplitudes in six NZs and six IZs after the formation of whole-cell recording (WCR) configuration. Currents were monitored by peak tail currents at −50 mV following 5-s pulses to +30 mV. (C) and (D) Current–voltage relationships of test pulse current (It) and tail current (Itail) in NZs and IZs. Same voltage clamp protocol as in (A). It was measured as the difference between the current level at the beginning of a voltage step and that at the end of the 5-s pulse. Itail was measured from the peak tail currents relative to the holding current. Currents in (B)–(D) are normalized by cell capacitance (pA/pF). Numbers of cells and hearts studied are shown. *P<0.01, NZs vs. IZs. Fig. 1 Open in new tabDownload slide (A) Currents from an NZ and an IZ before (control) and after addition of azimilide. Inset: voltage clamp protocol, holding voltage (Vh) −50 mV, test voltages (Vt) −40 to +70 mV in 10 mV increments for 5 s once every 30 s. Doted lines: zero current level. Arrowheads: step increase in current amplitude upon depolarization to +70 mV. (B) Time course of changes in delayed rectifier current amplitudes in six NZs and six IZs after the formation of whole-cell recording (WCR) configuration. Currents were monitored by peak tail currents at −50 mV following 5-s pulses to +30 mV. (C) and (D) Current–voltage relationships of test pulse current (It) and tail current (Itail) in NZs and IZs. Same voltage clamp protocol as in (A). It was measured as the difference between the current level at the beginning of a voltage step and that at the end of the 5-s pulse. Itail was measured from the peak tail currents relative to the holding current. Currents in (B)–(D) are normalized by cell capacitance (pA/pF). Numbers of cells and hearts studied are shown. *P<0.01, NZs vs. IZs. 2.2.3 Data acquisition and analysis Clamp protocol generation and data acquisition were controlled by Clampex (version 5.5 or 6), via a D/A and A/D converter (Digidata 1200 or DMA, Axon Instruments). Membrane currents were low-pass filtered at 1 kHz (Frequency Devices, Harverhill, MA) and digitized at a sampling interval of 5 ms. Methods of data analysis will be described in figure legends. Clampfit of pClamp (version 6.04) was used for amplitude measurement and curve fitting. 2.2.4 Experimental solutions The pipette solution contained (mM): KOH 115, aspartic acid 115, EGTA 10, HEPES 10, dextrose 5, ATP (K salt) 5, MgCl2 1, pH 7.3 with KOH. Normal Tyrode's solution had (mM): NaCl 146, KCl 4, CaCl2 2, MgCl2 0.5, HEPES 5, dextrose 5.5 (pH 7.3 with NaOH). The nominally Na- and Ca-free solution contained (mM): choline Cl 146, MgCl2 2.5, HEPES 5, dextrose 5.5, and nisoldipine 0.001. The pH of this solution was titrated to 7.3 with KOH, and KCl was added to make the final [K] equal 4 mM. 2.3 RNase protection assay (RPA) The 28S rRNA (28S) antisense probe (Ambion, Dallas, TX) was against a sequence conserved between human, rat, mouse and Xenopus (nucleotides 4400–4515 of GenBank Accession # M11167). Partial clones of dKvLQT1, dIsK and dERG were obtained from normal canine ventricular RNA by RT-PCR, and used as templates to make antisense probes in the presence of α-32P-UTP. Total RNAs were prepared from tissues using the acid guanidinium thiocyanate phenol–chloroform extraction method [11]. RPA was carried out using a commercial kit (RPA II, Ambion) according to the manufacturer's suggestions. Thirty ug of total RNA isolated from each sample was hybridized to radioactive antisense probes for 28S and one of the three channel subunits (dKvLQT1, dIsK or dERG). Thirty μg of yeast RNA was used as a negative control for probe self-protection bands. The gel was exposed to a PhosphoImager screen and the band intensities were quantified (model 445 SI, Molecular Dynamics). Each gel contained 4 lanes of samples (RNase digestion products) from control hearts and 4 lanes of samples from infarcted hearts (each sample from an individual animal) (see Figs. 4–6). To correct for differences in band intensity between lanes caused by sample handling and loading, the signal from the channel subunit in each lane was normalized by that from 28S (internal control) in the same lane. This is defined as ‘28S ratio’. Since radioactive probes used in different gels were synthesized in separate reactions and had different specific activities, a second normalization procedure was required to allow data to be pooled from different gels. This was done by normalizing data (values of 28S ratio) with the mean value of 28S ratio from the four control hearts in each gel. 2.4 Statistical analysis When appropriate, data are presented as mean±SE. Statistical analysis was performed using SigmaStat (version 2.1, Jandel Scientific): ANOVA multiple-group comparison followed by Tukey pair-wise comparison or Student's t-test. 3 Results 3.1 A. Delayed rectifier current density is reduced in canine ventricular myocytes from infarcted hearts Current traces recorded from an NZ are shown in Fig. 1A. The recording conditions were designed to eliminate interfering currents: Na and Ca currents were suppressed by the nominally Na- and Ca-free bath solution. Interference by the transient outward current (Ito) was minimized by the depolarized holding voltage (−50 mV, ∼50% Ito inactivation) and suppression by nisoldipine (1 μM) [12]. Depolarization pulses to−20 mV elicited time-dependent outward currents that continued to increase during the 5-s pulses. Subsequent repolarization to −50 mV was accompanied by slowly decaying outward tail currents. These are hallmarks of delayed rectifier currents in cardiac myocytes [4,5]. Indeed, both the time-dependent outward currents during depolarization and the subsequent tail currents upon repolarization were suppressed by azimilide (10 μM, Fig. 1A). At this concentration, azimilide blocks IKr and IKs with little or no effects on other K channels in canine myocytes [13]. Under the same conditions, the same voltage clamp protocol elicited much smaller time-dependent outward currents in the IZ than those of the NZ (Fig. 1A). Furthermore, there were no clear tail currents upon repolarization. In both NZs and IZs, membrane depolarization to >+20 mV induced a step increase in outward current. The magnitude of this time-independent current did not differ between the two. This current component was not suppressed by azimilide (NZ in Fig. 1A), but often increased with time (IZ in Fig. 1A, NZ in Fig. 2A). The identity of this current is not clear, and it is not considered in this study. Fig. 2 Open in new tabDownload slide Separation of IKr and IKs. Inset: voltage clamp protocol, Vh −50 mV, Vt −10 or +50 mV for 5 s, followed by repolarization to −50 mV. (A) Currents from an NZ under the control conditions (left), and in the presence of dofetilide (middle) or azimilide (right). Thin traces denote currents induced by Vt to −10 mV, and thick traces are those induced by Vt to +50 mV. Tail currents recorded under the control conditions (marked by the shaded area) were used for the quantification of IKr and IKs in (B). Dotted line denote zero current level. (B) Control tail currents from (A) are shown on expanded scales. The tail current following Vt to −10 mV reflects mainly IKr, and that following Vt to +50 mV includes both IKr and IKs. The IKs component is obtained by subtracting the tail current recorded after Vt to −10 mV from that after Vt to +50 mV (middle panel). IKr and IKs amplitudes were quantified from the peak tail currents vs. reference (dotted line). (C) Comparison of current densities of isolated IKr and IKs between NZs and IZs. Data were from 19 normal hearts and 15 infarcted hearts. Each heart had one IKr and one IKs current density averaged from data from that particular heart (1 to 6 cells per heart). Fig. 2 Open in new tabDownload slide Separation of IKr and IKs. Inset: voltage clamp protocol, Vh −50 mV, Vt −10 or +50 mV for 5 s, followed by repolarization to −50 mV. (A) Currents from an NZ under the control conditions (left), and in the presence of dofetilide (middle) or azimilide (right). Thin traces denote currents induced by Vt to −10 mV, and thick traces are those induced by Vt to +50 mV. Tail currents recorded under the control conditions (marked by the shaded area) were used for the quantification of IKr and IKs in (B). Dotted line denote zero current level. (B) Control tail currents from (A) are shown on expanded scales. The tail current following Vt to −10 mV reflects mainly IKr, and that following Vt to +50 mV includes both IKr and IKs. The IKs component is obtained by subtracting the tail current recorded after Vt to −10 mV from that after Vt to +50 mV (middle panel). IKr and IKs amplitudes were quantified from the peak tail currents vs. reference (dotted line). (C) Comparison of current densities of isolated IKr and IKs between NZs and IZs. Data were from 19 normal hearts and 15 infarcted hearts. Each heart had one IKr and one IKs current density averaged from data from that particular heart (1 to 6 cells per heart). Delayed rectifier currents, in particular the IKs component, tend to ‘run down’ in isolated myocytes. To minimize this interference, we began data collection 12–13 min after the beginning of whole-cell recording (WCR, see Methods). The voltage clamp protocol (shown in Fig. 1A) required 6 min to complete. Therefore, the duration of data collection was 12 to 19 min after the beginning of WCR. During this window of time, the current amplitudes were relatively stable, as illustrated in Fig. 1B (shaded area). Note that the separation between NZ and IZ data points is clear, and thus ‘run-down’ can not account for differences observed. However, prolonged (<20–25 min) intracellular dialysis was accompanied by a run down of current amplitudes in both NZs and IZs (data not shown). The amplitudes of delayed rectifier currents in NZs and IZs were quantified by the time-dependent outward currents observed during 5-s depolarization pulses (test pulse currents or It) and by the peak amplitudes of tail currents (Itail) recorded at −50 mV. Since there was a significant increase in the cell capacitance in IZs relative to NZs (215.6±9.7 vs. 172.4±7.3 pF, P<0.001) [2], each current amplitude was normalized by the cell's capacitance for current density. As shown in Fig. 1C and 1D, current densities of both It and Itail were reduced in IZs versus NZs. 3.2 B. Both IKr and IKs are reduced in myocytes from 5 day EBZ There are two components of delayed rectifier current in canine ventricular myocytes: the rapid (IKr) and the slow (IKs) components [4,5]. To determine whether changes in either or both contributed to the observed reduction in total delayed rectifier current densities in IZs, we sought to separate the two. In preliminary experiments, we attempted to separate the two by using an IKr-specific blocker (dofetilide) [5]. However, this attempt was hampered by the problem of IKs run-down, since we found that the drug-sensitive current was not pure IKr but also contained a component of ‘run-down’ IKs. Therefore, we used another approach. Under our recording conditions (nominally Nao- and Cao-free), IKr was activated in a more negative voltage range (−30 to −10 mV) than that of IKs (≥ 0 mV) [5]. IKr displayed an inward rectification (reduction of outward currents at strong depolarized voltages >+40 mV) but outward IKs currents continued to increase up to +70 mV [5]. Fig. 2A illustrates the distinction between IKr and IKs in an NZ. A depolarization pulse to −10 mV elicited a small time-dependent outward current, followed by a slowly decaying tail current upon repolarization to −50 mV. Both were suppressed by dofetilide (1 μM, middle panel of Fig. 2A), confirming that this low voltage depolarization mainly activated IKr. A strong depolarization pulse to +50 mV elicited a much larger time-dependent outward current. This was followed by a tail current that was larger and decayed faster than the tail current following Vt to −10 mV. Note that dofetilide had little effect on the time-dependent outward current at +50 mV, illustrating that IKr contributed little to currents at Vt≥ +40 mV due to inward rectification. Dofetilide reduced the tail current amplitude following Vt to +50 mV, as was expected for the suppression of the IKr component. The dofetilide-resistant current component was suppressed by azimilide (5 μM, right panel of Fig. 2A), confirming that IKs was mainly responsible for this component. The peak amplitude of tail current following Vt to −10 mV was used as a measure of IKr (Fig. 2B, left). Using this to approximate the IKr component of the tail current following Vt to +50 mV (IKr activation plateaued between −10 and +50 mV [4,5]), the difference current obtained by subtracting the tail current following Vt to −10 mV (IKr) from that following Vt to +50 mV (including both IKr and IKs) was used as a measure of IKs (Fig. 2B, right). As shown in Fig. 2C, both the IKr and IKs current densities were reduced in IZs versus NZs. 3.3 C. The IKr and IKs kinetics are altered in myocytes from 5 day EBZ The role of delayed rectifier currents in action potential repolarization depends on not only the fully-activated current amplitude, but also the kinetics of current activation and deactivation. We examined the kinetic properties of IKr and IKs in NZs and IZs. The analysis was limited to −10 and +50 mV at which IKr and IKs could be separated (Fig. 3 legend). In both NZs and IZs the activation and deactivation of IKr could be well described by a single exponential function (Fig. 3A, left panels). The rate of IKr activation at −10 mV was significantly faster in IZs (τ = 2.8±0.4 s) than in NZs (τ = 4.6±0.7 s) (Fig. 3B). On the other hand, the IKr deactivation rate at −50 mV was similar in NZs and IZs (τ = 1.23±0.09 and 1.15±0.15 s, respectively). Fig. 3 Open in new tabDownload slide Comparison of IKr and IKs kinetics in NZs and IZs. (A) Examples of curve fitting to estimate time constants (τ) of activation and deactivation. Top: voltage clamp protocol, Vh −50 mV, Vt −10 (left) or +50 (right) mV for 5 s followed by repolarization to −50 mV. Currents during depolarization pulses and tail currents following Vt to −10 mV were fit with a single exponential function. Tail currents following Vt to +50 mV were fit with a double exponential function. Data points are superimposed on curves calculated with best-fit parameter values. For the sake of clarity, only every 10th data points are shown. Dotted lines denote zero current level. (B) Summary of τ values of activation at −10 mV (representing IKr) and +50 mV (representing IKs) and τ values of deactivation at −50 mV following Vt to −10 mV (IKr deactivation) or +50 mV (IKs deactivation, corresponding to the fast component of τ). Numbers in bars denote those of cells and hearts studied. Fig. 3 Open in new tabDownload slide Comparison of IKr and IKs kinetics in NZs and IZs. (A) Examples of curve fitting to estimate time constants (τ) of activation and deactivation. Top: voltage clamp protocol, Vh −50 mV, Vt −10 (left) or +50 (right) mV for 5 s followed by repolarization to −50 mV. Currents during depolarization pulses and tail currents following Vt to −10 mV were fit with a single exponential function. Tail currents following Vt to +50 mV were fit with a double exponential function. Data points are superimposed on curves calculated with best-fit parameter values. For the sake of clarity, only every 10th data points are shown. Dotted lines denote zero current level. (B) Summary of τ values of activation at −10 mV (representing IKr) and +50 mV (representing IKs) and τ values of deactivation at −50 mV following Vt to −10 mV (IKr deactivation) or +50 mV (IKs deactivation, corresponding to the fast component of τ). Numbers in bars denote those of cells and hearts studied. The kinetics of IKs activation were approximated from the time-dependent outward currents recorded at +50 mV, when the contribution of IKr was small due to its inward rectification. As illustrated in the right panels of Fig. 3A, this current component in both NZs and IZs could be well described by a single exponential function. The rate of IKs activation in IZs was slower than that in NZs (τ = 9.1±3.9 vs. 4.6±0.6 s), although the difference was not statistically significant. The tail currents following Vt to +50 mV were fit with a double exponential function, and the fast component corresponded to the deactivation of IKs (Fig. 2B) [4]. The apparent rate of IKs deactivation at –50 mV was significantly faster in IZs than in NZ s (τ = 0.35±0.04 vs. 0.46±0.04 s) (Fig. 3B). 3.4 D. The mRNA levels of IKr and IKs channel subunits are reduced in EBZ but the changes are not similar among the subunits NZs and IZs might differ in their sensitivity to the cell isolation procedure and this could affect the amplitudes of currents measured [14]. Furthermore, it is possible that the changes in current density and kinetics are due to infarction-associated alterations in posttranslational modifications of IKr and IKs channels, with no changes in the number of channel proteins. To test whether these possibilities could account for the observed changes in current densities, we compared the mRNA levels of putative IKr and IKs subunits (dERG, dKvLQT1 and dIsK) [8–10,15]. A decrease in the level of pore-forming subunits (dERG and dKvLQT1) and/or a reduction of dIsK expression would suppress the current amplitudes [9,10,15]. RNA samples were prepared from infarct zone tissues 2 and 5 days after LAD occlusion, and from the corresponding region of control hearts (‘LV’ of control, 2-D and 5-D infarct in Figs. 4–7). Furthermore, to test whether the changes were specifically related to myocardial infarction, RNA samples were also prepared from right ventricular tissue (‘RV’ in Figs. 4–7) and used in RNase protection assays. Fig. 4 Open in new tabDownload slide Quantification of dKvLQT1 mRNA levels using the RNase protection assay. Shown are gel images from four experiments examining the dKvLQT1 mRNA levels in left ventricles (LV, top row) or right ventricles (RV, bottom row) of control hearts (control) or infarcted hearts two days (2-D infarct, left column) or five days (5-D infarct, right column) after LAD occlusion. On each gel, lanes 3 to 10 contained RNase digestion products with tissue types labeled on top. Lane 1 contained RNA size markers (100–500 nt in 100 nt increments). Lane 2 of gels on the left contained probes (dKvLQT1: 399 nt, 28S rRNA: 123 nt). Lanes 2 of gels on the right contained RNase digested yeast RNA (30 μg) as a negative control. Marked on the right are positions of probes (open symbols) and protected fragments (filled symbols). Fig. 4 Open in new tabDownload slide Quantification of dKvLQT1 mRNA levels using the RNase protection assay. Shown are gel images from four experiments examining the dKvLQT1 mRNA levels in left ventricles (LV, top row) or right ventricles (RV, bottom row) of control hearts (control) or infarcted hearts two days (2-D infarct, left column) or five days (5-D infarct, right column) after LAD occlusion. On each gel, lanes 3 to 10 contained RNase digestion products with tissue types labeled on top. Lane 1 contained RNA size markers (100–500 nt in 100 nt increments). Lane 2 of gels on the left contained probes (dKvLQT1: 399 nt, 28S rRNA: 123 nt). Lanes 2 of gels on the right contained RNase digested yeast RNA (30 μg) as a negative control. Marked on the right are positions of probes (open symbols) and protected fragments (filled symbols). Fig. 5 Open in new tabDownload slide Quantification of dIsK mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 5 Open in new tabDownload slide Quantification of dIsK mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 6 Open in new tabDownload slide Quantification of dERG mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 6 Open in new tabDownload slide Quantification of dERG mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 7 Open in new tabDownload slide Quantification of mRNA levels of dKvLQT1 (top), dIsK (middle) and dERG (bottom) in left ventricles (LV, left column) or right ventricles (RV, right column) of control hearts (open bars), or infarcted hearts 2 days (2-D infarct, shaded bars) or 5 days (5-D infarct, filled bars) after LAD occlusion Each group included eight hearts. *P<0.01 infarct vs. control. Fig. 7 Open in new tabDownload slide Quantification of mRNA levels of dKvLQT1 (top), dIsK (middle) and dERG (bottom) in left ventricles (LV, left column) or right ventricles (RV, right column) of control hearts (open bars), or infarcted hearts 2 days (2-D infarct, shaded bars) or 5 days (5-D infarct, filled bars) after LAD occlusion Each group included eight hearts. *P<0.01 infarct vs. control. Fig. 4 illustrates data from four experiments examining the mRNA levels of dKvLQT1 in LV and RV of control and infarcted hearts. The same format is used to present data obtained for dIsK (Fig. 5) and dERG (Fig. 6). Data are summarized in Fig. 7. In LV of 2-D infarcts, the mRNA levels of all three channel subunits were markedly reduced (to 55±4%, 32±5%, and 52±9% of control for dKvLQT1, dIsK and dERG). On the other hand, there were no significant changes in the RV mRNA levels by day 2, suggesting that the changes in LV were specifically related to the infarction process. By day 5 after LAD occlusion, the mRNA levels of dIsK and dERG in LV were still reduced (to 24±6% and 48±7% of control, respectively), although that of dKvLQT1 returned to the control level (107±6%). In RV, there was a reduction of dIsK mRNA (to 56±7% of control) with no significant changes in dKvLQT1 or dERG. These observations suggest that the mRNA level of dIsK was subject to down regulation by factors in addition to infarction per se that had a delayed onset. 4 Discussion The major findings of this study can be summarized as follows: (1) in myocytes isolated from the EBZ overlying a 5-day old infarct, the current densities of both IKr and IKs were reduced, (2) kinetic analysis suggest that there were accompanying changes in the current kinetics: the rate of IKr activation (measured at −10 mV) and that of IKs deactivation (measured at −50 mV) were faster in IZs than in NZs, (3) 2 days after LAD occlusion, the mRNA levels of three channel subunits that form the IKr (dERG and dIsK) and IKs (dKvLQT1 and dIsK) channels were reduced in infarct zone tissue. By day 5, the mRNA levels of dERG and dIsK remained reduced but that of dKvLQT1 returned to the control level, and (4) in non-infarcted right ventricle there was also a reduction in dIsK mRNA level 5 days after LAD occlusion, although no other changes in all three mRNAs by day 2 or in mRNAs of dKvLQT1 and dERG by day 5 were noted. 4.1 A. Technical considerations As previously described [2], IZs dispersed from the 5-day infarcted heart constituted a heterogeneous population, with many of them having a ruffled appearance and small dark droplets. Our previous reports have shown that cells with these morphologic characteristics manifest abnormal action potentials, similar to those recorded from multicellular tissue dissected from EBZ 4–5 days after LAD occlusion [1,2]. This indicates that these myocytes retained many of the electrophysiological abnormalities associated with the infarction process. We did not study cells from sham-operated hearts since we have previously reported that the electrical properties of these cells are not different from those from noninfarcted control hearts [2]. The determination of IKs activation time constant might be limited by the depolarization duration (5 s) in the voltage clamp protocols. Infarct zone myocytes in their native environment (in in situ heart) may be exposed to abnormalities in cellular milieu both intracellularly and extracellularly, such as alterations in ionic composition, cytoplasmic ATP level and enzymatic activities, and neurohumoral regulation [3]. The current densities and gating kinetics of both IKr and IKs are likely to be modulated by many of these factors. In our experiments, the currents were recorded under the whole-cell dialysis conditions that controlled both intracellular and extracellular milieu. Therefore, our observations most likely reflected ‘chronic’ changes in channel proteins resulting from alterations at the transcription and translation levels. 4.2 B. Relationship between changes in the mRNA levels of channel subunits and alterations in IKr and IKs current densities and kinetics Two days after LAD occlusion the mRNA levels of all three subunits for IKr and IKs channels were reduced. This might lead to a decrease in the translation of these proteins, and a decrease in the number of functional channels in infarct zone myocytes 5 days after LAD occlusion when cellular electrophysiology was studied. However, the observed changes in IKr and IKs kinetics could not be readily explained by a reduction in the number of functional channels. Alterations in the activities of enzymes such as protein kinases and phosphatases may affect the current kinetics. Another possibility is that these kinetic changes were due to alterations in the channel structure, which in turn might be caused by alterations in the subunit composition. Based on data from heterologous expression of IsK and KvLQT1 subunits in Xenopus oocytes and in mammalian cells, a high abundance of IsK subunit relative to that of KvLQT1 will increase the IKs current level and slow its activation [16,17]. It has been suggested that an association of IsK subunit with ERG can augment the IKr current level [9,18], and accelerate its deactivation [18]. Therefore, changes in the subunit composition, and in particular the abundance of IsK proteins, would have profound effects on the kinetics of IKr and IKs. Our RNase protection assays showed that the degree of reduction of dIsK mRNA exceeded those of dKvLQT1 and dERG mRNAs in infarct zone tissue. For example, two days after LAD occlusion the dIsK mRNA was reduced to 32±5% of control, while the dKvLQT1 and dERG mRNAs were reduced to 55±4% and 52±9% of control, respectively. By day 5, dIsK mRNA was reduced to 24±6%, while dERG was reduced to 48±7% and dKvLQT1 returned to control. If these led to a corresponding pattern of changes in the protein level, i.e. a decrease in the abundance of dIsK proteins relative to that of dERG and dKvLQT1, one would expect to see an acceleration of IKs activation and a slowing of IKr deactivation. Neither of these expected changes were observed in our experiments. Instead, we saw a slowing of IKs activation (although not statistically significant), accompanied by an acceleration of IKs deactivation at –50 mV. There was no change in the rate of IKr deactivation, although its activation at –10 mV was accelerated. The reasons for these observed kinetic changes and for the discrepancy between expectations and observations are not clear at present, but several explanations can be offered. First, the kinetic changes in IKr and IKs we observed 5 days after LAD occlusion might result from alterations in subunit transcripts that occurred after day 2, and the functional consequence of changes in subunit transcripts we saw by day 5 would be manifested at a later time. Second, there may be other subunits in canine ventricular myocytes that can regulate the gating behavior of IKr and IKs channels. Recently, a family of IsK (or MinK)-related Proteins (MiRPs) has been identified in human and mouse heart [19]. It is suggested that these proteins are able to be associated with KvLQT1 and/or ERG subunits and modify their channel function. At lease one MiRP isoform is present in canine ventricle (authors' unpublished results), and it may be subject to modulation by the infarction process. Third, changes in the mRNA level may not reflect changes in functional subunits because infarction may affect many processes such as protein translation, subunit assembly, processing, and trafficking. Future experiments should be directed toward measuring the protein levels of different subunits of IKr and IKs channels in infarct zone myocytes at different time points after LAD occlusion, and exploring the role of MiRP isoforms in IKr and IKs channel function in canine ventricular myocytes. 4.3 C. Implications of the present findings for abnormalities in cellular and tissue electrophysiology associated with myocardial infarction In infarcted hearts, both IKr and IKs may continue to contribute to action potential repolarization in myocytes far from the infarct zone. However, their role in EBZ myocytes may be reduced or altered. Our data show that the IKs current density in 5 day IZs is reduced and its activation slowed. These changes, in conjunction with the depressed plateau voltage of action potentials in these myocytes [20], will diminish or abolish IKs activation. The acceleration of IKs deactivation will further limit the use-dependence of IKs contribution to repolarization during tachycardia. The role of IKr in action potential repolarization may be reduced due to its lower current density. However, this may be partially compensated by the acceleration of IKr activation as seen here at −10 mV. Therefore, IKr may become more important (relative to IKs) in action potential repolarization in IZs than in NZs. This may impact on the effects of changing heart rate, β-adrenergic tone and extracellular [K] on action potential duration in different regions of an infarcted heart, because IKr and IKs channels respond differently to these factors [21,22]. Our RNase protection assays showed that 5 days after LAD occlusion, in both infarct zone (LV) and noninfarct zone (RV) the dIsK mRNA was disproportionately reduced relative to those of the pore-forming subunits (dKvLQT1 and dERG). These suggest that the dIsK mRNA is subject to down-regulation by factors other than infarction per se that have a delayed onset. In view of the important role of IsK subunits in determining the current level, gating kinetics and pharmacology of IKs and IKr channels discussed above [10,15,18,23], this change in dIsK message may have significant functional consequences that are manifested at a later time in infarcted heart. Acknowledgements We thank Dr. Randy S. Wymore for his generous gift of the dERG probe. This work was supported by HL 30557 and HL 46451 from National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD. References [1] Ursell P.C Gardner P.I Albala A Fenoglio J.J Wit A.L Structural and electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing Circ Res 1985 56 436 451 Google Scholar Crossref Search ADS PubMed WorldCat [2] Lue W Boyden P.A Abnormal electrical properties of myocytes from chronically infarcted canine heart. Alterations in Vmax and the transient outward current Circulation 1992 85 1175 1188 Google Scholar Crossref Search ADS PubMed WorldCat [3] Janse M.J Wit A.L Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction Physiological Reviews 1989 69 1049 1169 Google Scholar PubMed OpenURL Placeholder Text WorldCat [4] Gintant G.A Two components of delayed rectifier current in canine atrium and ventricle. Does I Ks play a role in the reverse rate dependence of class III agents? Circ Res 1996 78 26 37 Google Scholar Crossref Search ADS PubMed WorldCat [5] Liu D.-W Antzelevitch C Characteristics of the delayed rectifier current (I Kr and I Ks) in canine ventricular epicardial, midmyocardial, and endocardial myocytes. A weaker I Ks contributes to the longer action potential of the M cell Circ Res 1995 76 351 365 Google Scholar Crossref Search ADS PubMed WorldCat [6] Wang Z Fermini B Nattel S Rapid and slow components of delayed rectifier current in human atrial myocytes Cardiov Res 1994 28 1540 1546 Google Scholar Crossref Search ADS WorldCat [7] Li G.-R Feng J Yue L Carrier M Nattel S Evidence for two components of delayed rectifier K+ current in human ventricular myocytes Circ Res 1996 78 689 696 Google Scholar Crossref Search ADS PubMed WorldCat [8] Sanguinetti M.C Jiang C Curran M.E Keating M.T A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I Kr potassium channel Cell 1995 81 299 307 Google Scholar Crossref Search ADS PubMed WorldCat [9] McDonald T.V Yu Z Ming Z et al. A minK-HERG complex regulates the cardiac potassium current I Kr Nature 1997 388 289 292 Google Scholar Crossref Search ADS PubMed WorldCat [10] Sanguinetti M.C Curran M.E Zou A et al. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac I Ks potassium channel Nature 1996 384 80 83 Google Scholar Crossref Search ADS PubMed WorldCat [11] Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction Anal Biochem 1987 162 156 159 Google Scholar Crossref Search ADS PubMed WorldCat [12] Gotoh Y Imaizumi Y Watanabe M et al. Inhibition of transient outward K current by DHP Ca2+ antagonists and agonists in rabbit cardiac myocytes Am J Physiol 1991 260 H1737 H1742 Google Scholar PubMed OpenURL Placeholder Text WorldCat [13] Yao J.-A Tseng G.-N Azmilide (NE-10064) can prolong or shorten the action potential duration in canine ventricular myocytes: Dependence on blockade of K. Ca, and Na channels J Cardiov Electrophysiol 1997 8 184 198 Google Scholar Crossref Search ADS WorldCat [14] Yue L Feng J Li G.-R Nattel S Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods Am J Physiol 1996 270 H2157 H2168 Google Scholar PubMed OpenURL Placeholder Text WorldCat [15] Barhanin J Lesage F Guillemare E et al. KvLQT1 and IsK (minK) proteins associate to form the I Ks cardiac potassium current Nature 1996 384 78 80 Google Scholar Crossref Search ADS PubMed WorldCat [16] Blumenthal E.M Kaczmarek L.K Modulation by cAMP of a slowly activating potassium channel expressed in xenopus oocytes J Neuroscience 1992 12 290 296 Google Scholar PubMed OpenURL Placeholder Text WorldCat [17] Cui J Kline R.P Pennefather P Cohen I.S Gating of IsK expressed in Xenopus oocytes depends on the amount of mRNA injected J Gen Physiol 1994 104 87 105 Google Scholar Crossref Search ADS PubMed WorldCat [18] Kupershmidt S Yang T Anderson M.E et al. Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system Circ Res 1999 84 146 152 Google Scholar Crossref Search ADS PubMed WorldCat [19] Abbott G.W Sesti F Splawski I et al. MiRP1 forms I Kr potassium channels with HERG and is associated with cardiac arrhythmia Cell 1999 97 175 187 Google Scholar Crossref Search ADS PubMed WorldCat [20] Boyden P.A Gardner P.I Wit A.L Action potentials of cardiac muscle in healing infarcts: the response to norepinephrine and caffeine J Molecular Cellular Cardiology 1988 20 525 537 Google Scholar Crossref Search ADS WorldCat [21] Sanguinetti M.C Jurkiewicz N.K Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents Pflugers Archiv 1992 420 180 186 Google Scholar Crossref Search ADS PubMed WorldCat [22] Sanguinetti M.C Jurkiewicz N.K Scott A Siegl P.K.S Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E4031 in guinea pig myocytes. Mechanism of action Circ Res 1991 68 77 84 Google Scholar Crossref Search ADS PubMed WorldCat [23] Busch A.E Busch G.L Ford E et al. The role of the IsK protein in the specific pharmacological properties of the I Ks channel comples Br J Pharmacol 1997 122 187 189 Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2000, European Society of Cardiology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cardiovascular Research Oxford University Press

Delayed rectifier K currents have reduced amplitudes and altered kinetics in myocytes from infarcted canine ventricle

Download PDF
10 pages

Loading next page...
 
/lp/oxford-university-press/delayed-rectifier-k-currents-have-reduced-amplitudes-and-altered-IrDNAylmaq

References (24)

Publisher
Oxford University Press
Copyright
Copyright © 2000, European Society of Cardiology
ISSN
0008-6363
eISSN
1755-3245
DOI
10.1016/S0008-6363(00)00159-0
Publisher site
See Article on Publisher Site

Abstract

Abstract Objective: The rapid (IKr) and slow (IKs) components of delayed rectifier currents play an important role in determining the cardiac action potential configuration. Abnormalities in their function may contribute to arrhythmogenesis under pathological conditions. We studied the effects of myocardial infarction on IKr and IKs in canine ventricular myocytes and their molecular basis. Methods: Infarct zone myocytes (IZs) were isolated from a thin layer of surviving epicardium overlying an infarct 5 days after a total occlusion of the left anterior descending (LAD) coronary artery. Normal myocytes (NZs) were isolated from the corresponding region of control hearts for comparison. Currents were recorded under the whole-cell patch clamp conditions. Results: Both IKr and IKs current densities were reduced in IZs versus NZs. Kinetic analysis further suggests an acceleration of IKr activation and IKs deactivation. RNase protection assays were used to quantify the mRNA levels of IKr and IKs channel subunits (dERG, dIsK and dKvLQT1) in tissue immediately adjacent to the region where myocytes were isolated. mRNA levels of all three subunits were reduced 2 days after LAD occlusion (by 48±9%, 68±5%, and 45±4% for dERG, dIsK and dKvLQT1, respectively, n = 8 each). By day 5, the dKvLQT1 message returned to control while those of dERG and dIsK remained reduced (by 52±7% and 76±6%, respectively). Conclusions: The decrease in IKr and IKs amplitudes and changes in their kinetics in infarcted tissue might be due to a decrease in functional channels and/or changes in their subunit composition. Heterogeneous changes in IKr and IKs in infarcted hearts may impact on the effects of varying heart rate or neurohumoral modulation on repolarization. K-channel, Myocytes, Infarction, Gene expression, Remodeling, Single channel currents Time for primary review 23 days. This article is referred to in the Editorial by M.W. Veldkamp (pages 11–22) in this issue. 1 Introduction A canine model of myocardial infarction (MI) created by total occlusion of the left anterior descending (LAD) coronary artery has been extensively studied [1,2]. This model exhibits many features of arrhythmias similar to observations of patients with MI [3]. Thus it is a useful model for studying mechanisms of arrhythmias post MI. In this model, 4 to 5 days after LAD occlusion there is a layer of surviving epicardium of varying thickness overlying the infarct (epicardial border zone or EBZ) [1]. Ventricular tachyarrhythmias induced by programmed stimulation often result from reentrant circuits formed within the EBZ [3]. Although perturbations in cellular electrophysiology of the EBZ have been well characterized and linked to arrhythmogenesis, there is an important omission in the previous studies: the effects of MI on the delayed rectifier currents have not been examined. There are two components of delayed rectifier currents: the rapid (IKr) and the slow (IKs) components [4,5]. Both have been described for human atrial and ventricular myocytes [6,7] and may participate in action potential repolarization, yet relatively little is known about how disease processes affect their function and expression. In this study, we quantified and characterized IKr and IKs in myocytes isolated from the EBZ (IZ) and from the corresponding region of normal hearts (NZ). In addition, we quantified the mRNA levels of three putative subunits of IKr and IKs channels (dERG, dIsK, and dKvLQT1, ‘d’ for ‘dog’ isoforms) [8–10]. dERG and dKvLQT1 are the pore-forming subunits [8,10], while dIsK (also called minK) is a regulatory subunit that can associate with dKvLQT1 or dERG and affect their channel function [9,10]. Our data showed that there was a reduction in the mRNA levels of these subunits in infarcted hearts, but the degree of reduction was not the same among the three. In particular, we observed an uneven change in dKvLQT1 and dIsK mRNA levels that may lead to alterations in IKs channel behavior (gating kinetics and pharmacology) manifested at a later time (<5 days) after LAD occlusion. 2 Methods 2.1 Surgery Experiments on animals were conducted in conformity with the Declaration of Helsinki (Br Med J 1964; ii: 177) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The surgical procedure was as described [2]. Two or five days later, the infarct zone tissue (EBZ) was removed for myocyte isolation and RNA preparation. Tissue from the corresponding region of normal hearts was used as a control. 2.2 Electrophysiology 2.2.1 Myocyte preparation Ventricular myocytes were enzymatically dispersed as previously described [2]. For NZs, only rod-shaped cells with clear striations and surface free of blebs were used for voltage clamp. IZs were chosen for study based on the following morphological criteria: a ruffled appearance, not very clear striations, irregular shape, and small dark droplets [2]. 2.2.2 Voltage clamp experiments Whole-cell currents were recorded using the conventional suction pipette method at 24–26°C. About 6 min after forming the whole-cell recording (WCR) configuration, the bath solution was switched to a nominally Na- and Ca-free solution for 6 min, after which data collection began (see Fig. 1B). Fig. 1 Open in new tabDownload slide (A) Currents from an NZ and an IZ before (control) and after addition of azimilide. Inset: voltage clamp protocol, holding voltage (Vh) −50 mV, test voltages (Vt) −40 to +70 mV in 10 mV increments for 5 s once every 30 s. Doted lines: zero current level. Arrowheads: step increase in current amplitude upon depolarization to +70 mV. (B) Time course of changes in delayed rectifier current amplitudes in six NZs and six IZs after the formation of whole-cell recording (WCR) configuration. Currents were monitored by peak tail currents at −50 mV following 5-s pulses to +30 mV. (C) and (D) Current–voltage relationships of test pulse current (It) and tail current (Itail) in NZs and IZs. Same voltage clamp protocol as in (A). It was measured as the difference between the current level at the beginning of a voltage step and that at the end of the 5-s pulse. Itail was measured from the peak tail currents relative to the holding current. Currents in (B)–(D) are normalized by cell capacitance (pA/pF). Numbers of cells and hearts studied are shown. *P<0.01, NZs vs. IZs. Fig. 1 Open in new tabDownload slide (A) Currents from an NZ and an IZ before (control) and after addition of azimilide. Inset: voltage clamp protocol, holding voltage (Vh) −50 mV, test voltages (Vt) −40 to +70 mV in 10 mV increments for 5 s once every 30 s. Doted lines: zero current level. Arrowheads: step increase in current amplitude upon depolarization to +70 mV. (B) Time course of changes in delayed rectifier current amplitudes in six NZs and six IZs after the formation of whole-cell recording (WCR) configuration. Currents were monitored by peak tail currents at −50 mV following 5-s pulses to +30 mV. (C) and (D) Current–voltage relationships of test pulse current (It) and tail current (Itail) in NZs and IZs. Same voltage clamp protocol as in (A). It was measured as the difference between the current level at the beginning of a voltage step and that at the end of the 5-s pulse. Itail was measured from the peak tail currents relative to the holding current. Currents in (B)–(D) are normalized by cell capacitance (pA/pF). Numbers of cells and hearts studied are shown. *P<0.01, NZs vs. IZs. 2.2.3 Data acquisition and analysis Clamp protocol generation and data acquisition were controlled by Clampex (version 5.5 or 6), via a D/A and A/D converter (Digidata 1200 or DMA, Axon Instruments). Membrane currents were low-pass filtered at 1 kHz (Frequency Devices, Harverhill, MA) and digitized at a sampling interval of 5 ms. Methods of data analysis will be described in figure legends. Clampfit of pClamp (version 6.04) was used for amplitude measurement and curve fitting. 2.2.4 Experimental solutions The pipette solution contained (mM): KOH 115, aspartic acid 115, EGTA 10, HEPES 10, dextrose 5, ATP (K salt) 5, MgCl2 1, pH 7.3 with KOH. Normal Tyrode's solution had (mM): NaCl 146, KCl 4, CaCl2 2, MgCl2 0.5, HEPES 5, dextrose 5.5 (pH 7.3 with NaOH). The nominally Na- and Ca-free solution contained (mM): choline Cl 146, MgCl2 2.5, HEPES 5, dextrose 5.5, and nisoldipine 0.001. The pH of this solution was titrated to 7.3 with KOH, and KCl was added to make the final [K] equal 4 mM. 2.3 RNase protection assay (RPA) The 28S rRNA (28S) antisense probe (Ambion, Dallas, TX) was against a sequence conserved between human, rat, mouse and Xenopus (nucleotides 4400–4515 of GenBank Accession # M11167). Partial clones of dKvLQT1, dIsK and dERG were obtained from normal canine ventricular RNA by RT-PCR, and used as templates to make antisense probes in the presence of α-32P-UTP. Total RNAs were prepared from tissues using the acid guanidinium thiocyanate phenol–chloroform extraction method [11]. RPA was carried out using a commercial kit (RPA II, Ambion) according to the manufacturer's suggestions. Thirty ug of total RNA isolated from each sample was hybridized to radioactive antisense probes for 28S and one of the three channel subunits (dKvLQT1, dIsK or dERG). Thirty μg of yeast RNA was used as a negative control for probe self-protection bands. The gel was exposed to a PhosphoImager screen and the band intensities were quantified (model 445 SI, Molecular Dynamics). Each gel contained 4 lanes of samples (RNase digestion products) from control hearts and 4 lanes of samples from infarcted hearts (each sample from an individual animal) (see Figs. 4–6). To correct for differences in band intensity between lanes caused by sample handling and loading, the signal from the channel subunit in each lane was normalized by that from 28S (internal control) in the same lane. This is defined as ‘28S ratio’. Since radioactive probes used in different gels were synthesized in separate reactions and had different specific activities, a second normalization procedure was required to allow data to be pooled from different gels. This was done by normalizing data (values of 28S ratio) with the mean value of 28S ratio from the four control hearts in each gel. 2.4 Statistical analysis When appropriate, data are presented as mean±SE. Statistical analysis was performed using SigmaStat (version 2.1, Jandel Scientific): ANOVA multiple-group comparison followed by Tukey pair-wise comparison or Student's t-test. 3 Results 3.1 A. Delayed rectifier current density is reduced in canine ventricular myocytes from infarcted hearts Current traces recorded from an NZ are shown in Fig. 1A. The recording conditions were designed to eliminate interfering currents: Na and Ca currents were suppressed by the nominally Na- and Ca-free bath solution. Interference by the transient outward current (Ito) was minimized by the depolarized holding voltage (−50 mV, ∼50% Ito inactivation) and suppression by nisoldipine (1 μM) [12]. Depolarization pulses to−20 mV elicited time-dependent outward currents that continued to increase during the 5-s pulses. Subsequent repolarization to −50 mV was accompanied by slowly decaying outward tail currents. These are hallmarks of delayed rectifier currents in cardiac myocytes [4,5]. Indeed, both the time-dependent outward currents during depolarization and the subsequent tail currents upon repolarization were suppressed by azimilide (10 μM, Fig. 1A). At this concentration, azimilide blocks IKr and IKs with little or no effects on other K channels in canine myocytes [13]. Under the same conditions, the same voltage clamp protocol elicited much smaller time-dependent outward currents in the IZ than those of the NZ (Fig. 1A). Furthermore, there were no clear tail currents upon repolarization. In both NZs and IZs, membrane depolarization to >+20 mV induced a step increase in outward current. The magnitude of this time-independent current did not differ between the two. This current component was not suppressed by azimilide (NZ in Fig. 1A), but often increased with time (IZ in Fig. 1A, NZ in Fig. 2A). The identity of this current is not clear, and it is not considered in this study. Fig. 2 Open in new tabDownload slide Separation of IKr and IKs. Inset: voltage clamp protocol, Vh −50 mV, Vt −10 or +50 mV for 5 s, followed by repolarization to −50 mV. (A) Currents from an NZ under the control conditions (left), and in the presence of dofetilide (middle) or azimilide (right). Thin traces denote currents induced by Vt to −10 mV, and thick traces are those induced by Vt to +50 mV. Tail currents recorded under the control conditions (marked by the shaded area) were used for the quantification of IKr and IKs in (B). Dotted line denote zero current level. (B) Control tail currents from (A) are shown on expanded scales. The tail current following Vt to −10 mV reflects mainly IKr, and that following Vt to +50 mV includes both IKr and IKs. The IKs component is obtained by subtracting the tail current recorded after Vt to −10 mV from that after Vt to +50 mV (middle panel). IKr and IKs amplitudes were quantified from the peak tail currents vs. reference (dotted line). (C) Comparison of current densities of isolated IKr and IKs between NZs and IZs. Data were from 19 normal hearts and 15 infarcted hearts. Each heart had one IKr and one IKs current density averaged from data from that particular heart (1 to 6 cells per heart). Fig. 2 Open in new tabDownload slide Separation of IKr and IKs. Inset: voltage clamp protocol, Vh −50 mV, Vt −10 or +50 mV for 5 s, followed by repolarization to −50 mV. (A) Currents from an NZ under the control conditions (left), and in the presence of dofetilide (middle) or azimilide (right). Thin traces denote currents induced by Vt to −10 mV, and thick traces are those induced by Vt to +50 mV. Tail currents recorded under the control conditions (marked by the shaded area) were used for the quantification of IKr and IKs in (B). Dotted line denote zero current level. (B) Control tail currents from (A) are shown on expanded scales. The tail current following Vt to −10 mV reflects mainly IKr, and that following Vt to +50 mV includes both IKr and IKs. The IKs component is obtained by subtracting the tail current recorded after Vt to −10 mV from that after Vt to +50 mV (middle panel). IKr and IKs amplitudes were quantified from the peak tail currents vs. reference (dotted line). (C) Comparison of current densities of isolated IKr and IKs between NZs and IZs. Data were from 19 normal hearts and 15 infarcted hearts. Each heart had one IKr and one IKs current density averaged from data from that particular heart (1 to 6 cells per heart). Delayed rectifier currents, in particular the IKs component, tend to ‘run down’ in isolated myocytes. To minimize this interference, we began data collection 12–13 min after the beginning of whole-cell recording (WCR, see Methods). The voltage clamp protocol (shown in Fig. 1A) required 6 min to complete. Therefore, the duration of data collection was 12 to 19 min after the beginning of WCR. During this window of time, the current amplitudes were relatively stable, as illustrated in Fig. 1B (shaded area). Note that the separation between NZ and IZ data points is clear, and thus ‘run-down’ can not account for differences observed. However, prolonged (<20–25 min) intracellular dialysis was accompanied by a run down of current amplitudes in both NZs and IZs (data not shown). The amplitudes of delayed rectifier currents in NZs and IZs were quantified by the time-dependent outward currents observed during 5-s depolarization pulses (test pulse currents or It) and by the peak amplitudes of tail currents (Itail) recorded at −50 mV. Since there was a significant increase in the cell capacitance in IZs relative to NZs (215.6±9.7 vs. 172.4±7.3 pF, P<0.001) [2], each current amplitude was normalized by the cell's capacitance for current density. As shown in Fig. 1C and 1D, current densities of both It and Itail were reduced in IZs versus NZs. 3.2 B. Both IKr and IKs are reduced in myocytes from 5 day EBZ There are two components of delayed rectifier current in canine ventricular myocytes: the rapid (IKr) and the slow (IKs) components [4,5]. To determine whether changes in either or both contributed to the observed reduction in total delayed rectifier current densities in IZs, we sought to separate the two. In preliminary experiments, we attempted to separate the two by using an IKr-specific blocker (dofetilide) [5]. However, this attempt was hampered by the problem of IKs run-down, since we found that the drug-sensitive current was not pure IKr but also contained a component of ‘run-down’ IKs. Therefore, we used another approach. Under our recording conditions (nominally Nao- and Cao-free), IKr was activated in a more negative voltage range (−30 to −10 mV) than that of IKs (≥ 0 mV) [5]. IKr displayed an inward rectification (reduction of outward currents at strong depolarized voltages >+40 mV) but outward IKs currents continued to increase up to +70 mV [5]. Fig. 2A illustrates the distinction between IKr and IKs in an NZ. A depolarization pulse to −10 mV elicited a small time-dependent outward current, followed by a slowly decaying tail current upon repolarization to −50 mV. Both were suppressed by dofetilide (1 μM, middle panel of Fig. 2A), confirming that this low voltage depolarization mainly activated IKr. A strong depolarization pulse to +50 mV elicited a much larger time-dependent outward current. This was followed by a tail current that was larger and decayed faster than the tail current following Vt to −10 mV. Note that dofetilide had little effect on the time-dependent outward current at +50 mV, illustrating that IKr contributed little to currents at Vt≥ +40 mV due to inward rectification. Dofetilide reduced the tail current amplitude following Vt to +50 mV, as was expected for the suppression of the IKr component. The dofetilide-resistant current component was suppressed by azimilide (5 μM, right panel of Fig. 2A), confirming that IKs was mainly responsible for this component. The peak amplitude of tail current following Vt to −10 mV was used as a measure of IKr (Fig. 2B, left). Using this to approximate the IKr component of the tail current following Vt to +50 mV (IKr activation plateaued between −10 and +50 mV [4,5]), the difference current obtained by subtracting the tail current following Vt to −10 mV (IKr) from that following Vt to +50 mV (including both IKr and IKs) was used as a measure of IKs (Fig. 2B, right). As shown in Fig. 2C, both the IKr and IKs current densities were reduced in IZs versus NZs. 3.3 C. The IKr and IKs kinetics are altered in myocytes from 5 day EBZ The role of delayed rectifier currents in action potential repolarization depends on not only the fully-activated current amplitude, but also the kinetics of current activation and deactivation. We examined the kinetic properties of IKr and IKs in NZs and IZs. The analysis was limited to −10 and +50 mV at which IKr and IKs could be separated (Fig. 3 legend). In both NZs and IZs the activation and deactivation of IKr could be well described by a single exponential function (Fig. 3A, left panels). The rate of IKr activation at −10 mV was significantly faster in IZs (τ = 2.8±0.4 s) than in NZs (τ = 4.6±0.7 s) (Fig. 3B). On the other hand, the IKr deactivation rate at −50 mV was similar in NZs and IZs (τ = 1.23±0.09 and 1.15±0.15 s, respectively). Fig. 3 Open in new tabDownload slide Comparison of IKr and IKs kinetics in NZs and IZs. (A) Examples of curve fitting to estimate time constants (τ) of activation and deactivation. Top: voltage clamp protocol, Vh −50 mV, Vt −10 (left) or +50 (right) mV for 5 s followed by repolarization to −50 mV. Currents during depolarization pulses and tail currents following Vt to −10 mV were fit with a single exponential function. Tail currents following Vt to +50 mV were fit with a double exponential function. Data points are superimposed on curves calculated with best-fit parameter values. For the sake of clarity, only every 10th data points are shown. Dotted lines denote zero current level. (B) Summary of τ values of activation at −10 mV (representing IKr) and +50 mV (representing IKs) and τ values of deactivation at −50 mV following Vt to −10 mV (IKr deactivation) or +50 mV (IKs deactivation, corresponding to the fast component of τ). Numbers in bars denote those of cells and hearts studied. Fig. 3 Open in new tabDownload slide Comparison of IKr and IKs kinetics in NZs and IZs. (A) Examples of curve fitting to estimate time constants (τ) of activation and deactivation. Top: voltage clamp protocol, Vh −50 mV, Vt −10 (left) or +50 (right) mV for 5 s followed by repolarization to −50 mV. Currents during depolarization pulses and tail currents following Vt to −10 mV were fit with a single exponential function. Tail currents following Vt to +50 mV were fit with a double exponential function. Data points are superimposed on curves calculated with best-fit parameter values. For the sake of clarity, only every 10th data points are shown. Dotted lines denote zero current level. (B) Summary of τ values of activation at −10 mV (representing IKr) and +50 mV (representing IKs) and τ values of deactivation at −50 mV following Vt to −10 mV (IKr deactivation) or +50 mV (IKs deactivation, corresponding to the fast component of τ). Numbers in bars denote those of cells and hearts studied. The kinetics of IKs activation were approximated from the time-dependent outward currents recorded at +50 mV, when the contribution of IKr was small due to its inward rectification. As illustrated in the right panels of Fig. 3A, this current component in both NZs and IZs could be well described by a single exponential function. The rate of IKs activation in IZs was slower than that in NZs (τ = 9.1±3.9 vs. 4.6±0.6 s), although the difference was not statistically significant. The tail currents following Vt to +50 mV were fit with a double exponential function, and the fast component corresponded to the deactivation of IKs (Fig. 2B) [4]. The apparent rate of IKs deactivation at –50 mV was significantly faster in IZs than in NZ s (τ = 0.35±0.04 vs. 0.46±0.04 s) (Fig. 3B). 3.4 D. The mRNA levels of IKr and IKs channel subunits are reduced in EBZ but the changes are not similar among the subunits NZs and IZs might differ in their sensitivity to the cell isolation procedure and this could affect the amplitudes of currents measured [14]. Furthermore, it is possible that the changes in current density and kinetics are due to infarction-associated alterations in posttranslational modifications of IKr and IKs channels, with no changes in the number of channel proteins. To test whether these possibilities could account for the observed changes in current densities, we compared the mRNA levels of putative IKr and IKs subunits (dERG, dKvLQT1 and dIsK) [8–10,15]. A decrease in the level of pore-forming subunits (dERG and dKvLQT1) and/or a reduction of dIsK expression would suppress the current amplitudes [9,10,15]. RNA samples were prepared from infarct zone tissues 2 and 5 days after LAD occlusion, and from the corresponding region of control hearts (‘LV’ of control, 2-D and 5-D infarct in Figs. 4–7). Furthermore, to test whether the changes were specifically related to myocardial infarction, RNA samples were also prepared from right ventricular tissue (‘RV’ in Figs. 4–7) and used in RNase protection assays. Fig. 4 Open in new tabDownload slide Quantification of dKvLQT1 mRNA levels using the RNase protection assay. Shown are gel images from four experiments examining the dKvLQT1 mRNA levels in left ventricles (LV, top row) or right ventricles (RV, bottom row) of control hearts (control) or infarcted hearts two days (2-D infarct, left column) or five days (5-D infarct, right column) after LAD occlusion. On each gel, lanes 3 to 10 contained RNase digestion products with tissue types labeled on top. Lane 1 contained RNA size markers (100–500 nt in 100 nt increments). Lane 2 of gels on the left contained probes (dKvLQT1: 399 nt, 28S rRNA: 123 nt). Lanes 2 of gels on the right contained RNase digested yeast RNA (30 μg) as a negative control. Marked on the right are positions of probes (open symbols) and protected fragments (filled symbols). Fig. 4 Open in new tabDownload slide Quantification of dKvLQT1 mRNA levels using the RNase protection assay. Shown are gel images from four experiments examining the dKvLQT1 mRNA levels in left ventricles (LV, top row) or right ventricles (RV, bottom row) of control hearts (control) or infarcted hearts two days (2-D infarct, left column) or five days (5-D infarct, right column) after LAD occlusion. On each gel, lanes 3 to 10 contained RNase digestion products with tissue types labeled on top. Lane 1 contained RNA size markers (100–500 nt in 100 nt increments). Lane 2 of gels on the left contained probes (dKvLQT1: 399 nt, 28S rRNA: 123 nt). Lanes 2 of gels on the right contained RNase digested yeast RNA (30 μg) as a negative control. Marked on the right are positions of probes (open symbols) and protected fragments (filled symbols). Fig. 5 Open in new tabDownload slide Quantification of dIsK mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 5 Open in new tabDownload slide Quantification of dIsK mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 6 Open in new tabDownload slide Quantification of dERG mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 6 Open in new tabDownload slide Quantification of dERG mRNA levels in LV and RV of control hearts and infarcted hearts 2 or 5 days after LAD occlusion. The format is the same as that of Fig. 4. Fig. 7 Open in new tabDownload slide Quantification of mRNA levels of dKvLQT1 (top), dIsK (middle) and dERG (bottom) in left ventricles (LV, left column) or right ventricles (RV, right column) of control hearts (open bars), or infarcted hearts 2 days (2-D infarct, shaded bars) or 5 days (5-D infarct, filled bars) after LAD occlusion Each group included eight hearts. *P<0.01 infarct vs. control. Fig. 7 Open in new tabDownload slide Quantification of mRNA levels of dKvLQT1 (top), dIsK (middle) and dERG (bottom) in left ventricles (LV, left column) or right ventricles (RV, right column) of control hearts (open bars), or infarcted hearts 2 days (2-D infarct, shaded bars) or 5 days (5-D infarct, filled bars) after LAD occlusion Each group included eight hearts. *P<0.01 infarct vs. control. Fig. 4 illustrates data from four experiments examining the mRNA levels of dKvLQT1 in LV and RV of control and infarcted hearts. The same format is used to present data obtained for dIsK (Fig. 5) and dERG (Fig. 6). Data are summarized in Fig. 7. In LV of 2-D infarcts, the mRNA levels of all three channel subunits were markedly reduced (to 55±4%, 32±5%, and 52±9% of control for dKvLQT1, dIsK and dERG). On the other hand, there were no significant changes in the RV mRNA levels by day 2, suggesting that the changes in LV were specifically related to the infarction process. By day 5 after LAD occlusion, the mRNA levels of dIsK and dERG in LV were still reduced (to 24±6% and 48±7% of control, respectively), although that of dKvLQT1 returned to the control level (107±6%). In RV, there was a reduction of dIsK mRNA (to 56±7% of control) with no significant changes in dKvLQT1 or dERG. These observations suggest that the mRNA level of dIsK was subject to down regulation by factors in addition to infarction per se that had a delayed onset. 4 Discussion The major findings of this study can be summarized as follows: (1) in myocytes isolated from the EBZ overlying a 5-day old infarct, the current densities of both IKr and IKs were reduced, (2) kinetic analysis suggest that there were accompanying changes in the current kinetics: the rate of IKr activation (measured at −10 mV) and that of IKs deactivation (measured at −50 mV) were faster in IZs than in NZs, (3) 2 days after LAD occlusion, the mRNA levels of three channel subunits that form the IKr (dERG and dIsK) and IKs (dKvLQT1 and dIsK) channels were reduced in infarct zone tissue. By day 5, the mRNA levels of dERG and dIsK remained reduced but that of dKvLQT1 returned to the control level, and (4) in non-infarcted right ventricle there was also a reduction in dIsK mRNA level 5 days after LAD occlusion, although no other changes in all three mRNAs by day 2 or in mRNAs of dKvLQT1 and dERG by day 5 were noted. 4.1 A. Technical considerations As previously described [2], IZs dispersed from the 5-day infarcted heart constituted a heterogeneous population, with many of them having a ruffled appearance and small dark droplets. Our previous reports have shown that cells with these morphologic characteristics manifest abnormal action potentials, similar to those recorded from multicellular tissue dissected from EBZ 4–5 days after LAD occlusion [1,2]. This indicates that these myocytes retained many of the electrophysiological abnormalities associated with the infarction process. We did not study cells from sham-operated hearts since we have previously reported that the electrical properties of these cells are not different from those from noninfarcted control hearts [2]. The determination of IKs activation time constant might be limited by the depolarization duration (5 s) in the voltage clamp protocols. Infarct zone myocytes in their native environment (in in situ heart) may be exposed to abnormalities in cellular milieu both intracellularly and extracellularly, such as alterations in ionic composition, cytoplasmic ATP level and enzymatic activities, and neurohumoral regulation [3]. The current densities and gating kinetics of both IKr and IKs are likely to be modulated by many of these factors. In our experiments, the currents were recorded under the whole-cell dialysis conditions that controlled both intracellular and extracellular milieu. Therefore, our observations most likely reflected ‘chronic’ changes in channel proteins resulting from alterations at the transcription and translation levels. 4.2 B. Relationship between changes in the mRNA levels of channel subunits and alterations in IKr and IKs current densities and kinetics Two days after LAD occlusion the mRNA levels of all three subunits for IKr and IKs channels were reduced. This might lead to a decrease in the translation of these proteins, and a decrease in the number of functional channels in infarct zone myocytes 5 days after LAD occlusion when cellular electrophysiology was studied. However, the observed changes in IKr and IKs kinetics could not be readily explained by a reduction in the number of functional channels. Alterations in the activities of enzymes such as protein kinases and phosphatases may affect the current kinetics. Another possibility is that these kinetic changes were due to alterations in the channel structure, which in turn might be caused by alterations in the subunit composition. Based on data from heterologous expression of IsK and KvLQT1 subunits in Xenopus oocytes and in mammalian cells, a high abundance of IsK subunit relative to that of KvLQT1 will increase the IKs current level and slow its activation [16,17]. It has been suggested that an association of IsK subunit with ERG can augment the IKr current level [9,18], and accelerate its deactivation [18]. Therefore, changes in the subunit composition, and in particular the abundance of IsK proteins, would have profound effects on the kinetics of IKr and IKs. Our RNase protection assays showed that the degree of reduction of dIsK mRNA exceeded those of dKvLQT1 and dERG mRNAs in infarct zone tissue. For example, two days after LAD occlusion the dIsK mRNA was reduced to 32±5% of control, while the dKvLQT1 and dERG mRNAs were reduced to 55±4% and 52±9% of control, respectively. By day 5, dIsK mRNA was reduced to 24±6%, while dERG was reduced to 48±7% and dKvLQT1 returned to control. If these led to a corresponding pattern of changes in the protein level, i.e. a decrease in the abundance of dIsK proteins relative to that of dERG and dKvLQT1, one would expect to see an acceleration of IKs activation and a slowing of IKr deactivation. Neither of these expected changes were observed in our experiments. Instead, we saw a slowing of IKs activation (although not statistically significant), accompanied by an acceleration of IKs deactivation at –50 mV. There was no change in the rate of IKr deactivation, although its activation at –10 mV was accelerated. The reasons for these observed kinetic changes and for the discrepancy between expectations and observations are not clear at present, but several explanations can be offered. First, the kinetic changes in IKr and IKs we observed 5 days after LAD occlusion might result from alterations in subunit transcripts that occurred after day 2, and the functional consequence of changes in subunit transcripts we saw by day 5 would be manifested at a later time. Second, there may be other subunits in canine ventricular myocytes that can regulate the gating behavior of IKr and IKs channels. Recently, a family of IsK (or MinK)-related Proteins (MiRPs) has been identified in human and mouse heart [19]. It is suggested that these proteins are able to be associated with KvLQT1 and/or ERG subunits and modify their channel function. At lease one MiRP isoform is present in canine ventricle (authors' unpublished results), and it may be subject to modulation by the infarction process. Third, changes in the mRNA level may not reflect changes in functional subunits because infarction may affect many processes such as protein translation, subunit assembly, processing, and trafficking. Future experiments should be directed toward measuring the protein levels of different subunits of IKr and IKs channels in infarct zone myocytes at different time points after LAD occlusion, and exploring the role of MiRP isoforms in IKr and IKs channel function in canine ventricular myocytes. 4.3 C. Implications of the present findings for abnormalities in cellular and tissue electrophysiology associated with myocardial infarction In infarcted hearts, both IKr and IKs may continue to contribute to action potential repolarization in myocytes far from the infarct zone. However, their role in EBZ myocytes may be reduced or altered. Our data show that the IKs current density in 5 day IZs is reduced and its activation slowed. These changes, in conjunction with the depressed plateau voltage of action potentials in these myocytes [20], will diminish or abolish IKs activation. The acceleration of IKs deactivation will further limit the use-dependence of IKs contribution to repolarization during tachycardia. The role of IKr in action potential repolarization may be reduced due to its lower current density. However, this may be partially compensated by the acceleration of IKr activation as seen here at −10 mV. Therefore, IKr may become more important (relative to IKs) in action potential repolarization in IZs than in NZs. This may impact on the effects of changing heart rate, β-adrenergic tone and extracellular [K] on action potential duration in different regions of an infarcted heart, because IKr and IKs channels respond differently to these factors [21,22]. Our RNase protection assays showed that 5 days after LAD occlusion, in both infarct zone (LV) and noninfarct zone (RV) the dIsK mRNA was disproportionately reduced relative to those of the pore-forming subunits (dKvLQT1 and dERG). These suggest that the dIsK mRNA is subject to down-regulation by factors other than infarction per se that have a delayed onset. In view of the important role of IsK subunits in determining the current level, gating kinetics and pharmacology of IKs and IKr channels discussed above [10,15,18,23], this change in dIsK message may have significant functional consequences that are manifested at a later time in infarcted heart. Acknowledgements We thank Dr. Randy S. Wymore for his generous gift of the dERG probe. This work was supported by HL 30557 and HL 46451 from National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD. References [1] Ursell P.C Gardner P.I Albala A Fenoglio J.J Wit A.L Structural and electrophysiological changes in the epicardial border zone of canine myocardial infarcts during infarct healing Circ Res 1985 56 436 451 Google Scholar Crossref Search ADS PubMed WorldCat [2] Lue W Boyden P.A Abnormal electrical properties of myocytes from chronically infarcted canine heart. Alterations in Vmax and the transient outward current Circulation 1992 85 1175 1188 Google Scholar Crossref Search ADS PubMed WorldCat [3] Janse M.J Wit A.L Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction Physiological Reviews 1989 69 1049 1169 Google Scholar PubMed OpenURL Placeholder Text WorldCat [4] Gintant G.A Two components of delayed rectifier current in canine atrium and ventricle. Does I Ks play a role in the reverse rate dependence of class III agents? Circ Res 1996 78 26 37 Google Scholar Crossref Search ADS PubMed WorldCat [5] Liu D.-W Antzelevitch C Characteristics of the delayed rectifier current (I Kr and I Ks) in canine ventricular epicardial, midmyocardial, and endocardial myocytes. A weaker I Ks contributes to the longer action potential of the M cell Circ Res 1995 76 351 365 Google Scholar Crossref Search ADS PubMed WorldCat [6] Wang Z Fermini B Nattel S Rapid and slow components of delayed rectifier current in human atrial myocytes Cardiov Res 1994 28 1540 1546 Google Scholar Crossref Search ADS WorldCat [7] Li G.-R Feng J Yue L Carrier M Nattel S Evidence for two components of delayed rectifier K+ current in human ventricular myocytes Circ Res 1996 78 689 696 Google Scholar Crossref Search ADS PubMed WorldCat [8] Sanguinetti M.C Jiang C Curran M.E Keating M.T A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I Kr potassium channel Cell 1995 81 299 307 Google Scholar Crossref Search ADS PubMed WorldCat [9] McDonald T.V Yu Z Ming Z et al. A minK-HERG complex regulates the cardiac potassium current I Kr Nature 1997 388 289 292 Google Scholar Crossref Search ADS PubMed WorldCat [10] Sanguinetti M.C Curran M.E Zou A et al. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac I Ks potassium channel Nature 1996 384 80 83 Google Scholar Crossref Search ADS PubMed WorldCat [11] Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction Anal Biochem 1987 162 156 159 Google Scholar Crossref Search ADS PubMed WorldCat [12] Gotoh Y Imaizumi Y Watanabe M et al. Inhibition of transient outward K current by DHP Ca2+ antagonists and agonists in rabbit cardiac myocytes Am J Physiol 1991 260 H1737 H1742 Google Scholar PubMed OpenURL Placeholder Text WorldCat [13] Yao J.-A Tseng G.-N Azmilide (NE-10064) can prolong or shorten the action potential duration in canine ventricular myocytes: Dependence on blockade of K. Ca, and Na channels J Cardiov Electrophysiol 1997 8 184 198 Google Scholar Crossref Search ADS WorldCat [14] Yue L Feng J Li G.-R Nattel S Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods Am J Physiol 1996 270 H2157 H2168 Google Scholar PubMed OpenURL Placeholder Text WorldCat [15] Barhanin J Lesage F Guillemare E et al. KvLQT1 and IsK (minK) proteins associate to form the I Ks cardiac potassium current Nature 1996 384 78 80 Google Scholar Crossref Search ADS PubMed WorldCat [16] Blumenthal E.M Kaczmarek L.K Modulation by cAMP of a slowly activating potassium channel expressed in xenopus oocytes J Neuroscience 1992 12 290 296 Google Scholar PubMed OpenURL Placeholder Text WorldCat [17] Cui J Kline R.P Pennefather P Cohen I.S Gating of IsK expressed in Xenopus oocytes depends on the amount of mRNA injected J Gen Physiol 1994 104 87 105 Google Scholar Crossref Search ADS PubMed WorldCat [18] Kupershmidt S Yang T Anderson M.E et al. Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system Circ Res 1999 84 146 152 Google Scholar Crossref Search ADS PubMed WorldCat [19] Abbott G.W Sesti F Splawski I et al. MiRP1 forms I Kr potassium channels with HERG and is associated with cardiac arrhythmia Cell 1999 97 175 187 Google Scholar Crossref Search ADS PubMed WorldCat [20] Boyden P.A Gardner P.I Wit A.L Action potentials of cardiac muscle in healing infarcts: the response to norepinephrine and caffeine J Molecular Cellular Cardiology 1988 20 525 537 Google Scholar Crossref Search ADS WorldCat [21] Sanguinetti M.C Jurkiewicz N.K Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents Pflugers Archiv 1992 420 180 186 Google Scholar Crossref Search ADS PubMed WorldCat [22] Sanguinetti M.C Jurkiewicz N.K Scott A Siegl P.K.S Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E4031 in guinea pig myocytes. Mechanism of action Circ Res 1991 68 77 84 Google Scholar Crossref Search ADS PubMed WorldCat [23] Busch A.E Busch G.L Ford E et al. The role of the IsK protein in the specific pharmacological properties of the I Ks channel comples Br J Pharmacol 1997 122 187 189 Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2000, European Society of Cardiology

Journal

Cardiovascular Research – Oxford University Press

Published: Oct 1, 2000

There are no references for this article.