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Cardiovascular Research 1999 44(1):132-145; doi:10.1016/S0008-6363(99)00154-6
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Heterogeneous changes in K currents in rat ventricles three days after myocardial infarction

Jian-An Yaoa, Min Jianga, Jing-Song Fanb, Ying-Ying Zhouc and Gea-Ny Tsenga,*

aDepartment of Pharmacology, Columbia University, New York, NY 10032, USA
bDepartment of Physiology and Biophysics, University of Texas, Medical Branch, Galveston, TX 77555-0641, USA
cLaboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224, USA

* Corresponding author. Tel.: +1-212-305-4166; fax: +1-212-305-8780 gt10{at}columbia.edu

Received 3 March 1999; accepted 12 April 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: After coronary artery occlusion, surviving myocardium in and around the infarct zone plays an important role in arrhythmogenesis. Understanding the mechanisms for derangements in cardiac electrical activity at the cellular and molecular levels is important for the design of effective therapeutic strategies. Methods: To provide part of that understanding, we studied changes in K channel function and expression in rat ventricular myocardium three days after occluding the left major coronary artery. The epicardium and endocardium of infarcted region in the left ventricle and the free wall of right ventricle were separated for myocyte isolation, followed by whole-cell voltage clamp studies. Myocytes were also isolated from corresponding regions of control and sham-operated hearts and studied under the same conditions. Results: We found that the transient outward (Ito), delayed rectifier (IK) and inward rectifier (IK1) currents have different distribution patterns in normal rat ventricular myocardium. Sham-operation did not affect any of these K currents in left ventricular myocytes, but coronary artery occlusion caused a reduction of all three. For Ito and IK1 the reduction was greater in epicardial than in endocardial myocytes, but IK was reduced equally in these two cell groups. Unexpectedly, Ito and IK as well as cell capacitance were increased in right ventricular myocytes from infarcted as well as sham-operated hearts. Western blot analysis indicated that the level of Kv4 channel proteins (Kv4.2+Kv4.3) was reduced in infarcted left ventricular myocardium, consistent with the reduction in Ito. Conclusion: Our data suggest that the distribution of K channels and changes in them induced by coronary artery occlusion are heterogeneous in ventricular myocardium. Understanding the molecular mechanisms for this heterogeneity and its implications in arrhythmogenesis poses a challenge in designing effective antiarrhythmic therapy for myocardial infarction patients.

KEYWORDS Rat; Kv4 subunits; Transient outward current; Myocardial infarction; K channel; Gene expression; Arrhythmias

This article is referred to in the Editorial by T. Kiyosue and M. Arita (pages 13–16) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
During the acute and subacute phases of myocardial infarction, surviving myocardium in and around an infarct zone plays an important role in arrhythmogenesis [1]. Information about the abnormalities in cellular electrophysiology and their molecular basis is important for an understanding of the mechanisms of arrhythmias occurring during this critical time after coronary artery occlusion. It is also crucial for the design of effective antiarrhythmic therapy.

Arrhythmias during the acute and subacute phases of myocardial infarction and abnormalities in cellular electrophysiology have been studied extensively in a canine model employing two-stage ligation of the left anterior descending coronary artery [1–9]. These studies indicate that changes in the action potential configuration in border zone tissues overlying an infarct can be largely accounted for by abnormalities in the function of Na, Ca and K channels and in intracellular Ca handling. The changes in action potential configuration, along with perturbations in intercellular coupling [3,10], contribute to the initiation and perpetuation of reentrant arrhythmias that involve infarct and border zone tissues. However, the molecular basis for these abnormalities in ion channel function, Cai handling, and intercellular coupling is largely unknown.

The purpose of the present study was to examine alterations in the function of K channels in rat ventricular myocytes during the subacute phase of myocardial infarction (3 days after coronary artery occlusion). We focused on three major K channels, two of which are important for determining the action potential configuration (transient outward channel, Ito, and delayed rectifier channel, IK) and one for maintaining the resting membrane potential (inward rectifier channel, IK1) [11,12]. We separated the epicardial and endocardial halves of left ventricles and the free wall of right ventricles from control, sham-operated and coronary artery-ligated hearts for myocyte isolation, and studied their K currents under the same conditions. A careful separation of myocytes derived from these sites is important because a transmural gradient of Ito between epicardium and endocardium of left ventricle and inhomogeneity in the expression of K channels between left and right ventricles have been described for rat and other species, including dog and human [13–19]. Furthermore, spatial inhomogeneity in the modulation of K channel function by disease conditions of the heart has been reported for rat, dog and human [18,20,21]. There were three goals in these electrophysiological experiments: (1) to study the distribution of current density of these three K channels in normal rat ventricles, (2) to examine how myocardial infarction and sham operation affected these K channels, and (3) to see whether the observed changes displayed any spatial inhomogeneity. These electrophysiological experiments showed that the current that was most profoundly affected by myocardial infarction was Ito. We further performed Western blot analysis to show that in the infarcted left ventricle there was an accompanying reduction in the protein level of the major {alpha} subunits underlying Ito in rat heart (Kv4.2 and Kv4.3) [22–24].


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Surgery
The investigation conforms with 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). Male Wistar rats weighing 325–350 g at the time of surgery were used. Myocardial infarction was created using sterile procedures. Under isoflurane anesthesia, the rat was intubated and ventilated. After left thoracotomy, the heart was exposed. The left major coronary artery was occluded approximately 2 mm from the tip of the left atrial appendage using 5.0 silk. If the heart was to be used for myocyte isolation, a small piece of thin polyethylene (PE) tubing (diameter ~0.5 mm, length <3 mm) was placed parallel to the coronary artery and ligated along with the artery. Prior to cell isolation (described below), this PE tubing was removed, allowing the previously occluded artery to open. Therefore the infarct zone could be perfused with a collagenase solution and digested. After the ligation, the chest was closed in layers and the animal was allowed to recover from anesthesia. About 60% of the animals survived the surgery. The sham operation was similar but did not include coronary artery ligation.

2.2 Ventricular myocyte preparation
Three days after the surgery, rats were anesthetized. Hearts were quickly excised and placed in Tyrode’s solution (composition given below). The infarct zone could be readily identified visually as a pale area in the antero-apical region of the left ventricle. The PE tubing was removed. After cannulation of the aorta, the heart was mounted on a Langendorff apparatus and perfused with 35 ml of Ca-free Tyrode’s solution, followed by a 10-min perfusion with the same solution containing collagenase (0.85 g/ml, type II, Worthington Biochemical, NJ) and trypsin (1 mg/ml, Gibco) at a pressure of 80 mm Hg. All solutions were equilibrated with 5% CO2/95% O2 and maintained at 34–36°C. At the end of enzyme perfusion, the infarct zone was softened (digested) as was the rest of the heart. The atria were removed. The infarct zone (previously identified visually as described above) and the adjacent region were isolated from the left ventricle and dissected into outer (epicardial) and inner (endocardial) halves. The free wall of the right ventricle was also isolated. The tissues were placed in separate petri dishes. They were minced and gently triturated for 15 min to release single myocytes. The supernatant was collected and the cells were pelleted by low-speed centrifugation. Cell pellets were resuspended in Tyrode’s solution containing 0.5 mM Ca and supplemented with insulin (4 µg/ml), mannitol (0.92 mg/ml) and pyruvate (0.5 mg/ml). The remaining tissue pieces were subject to further trituration for 3 to 5 more cycles. Myocytes were also prepared from corresponding regions of control and sham-operated hearts. Myocytes were kept in an incubator of 5% CO2/air at 37°C, and used within 12 h after isolation.

2.3 Electrophysiological experiments
Whole-cell currents were recorded using the conventional suction pipette method [25] with an Axopatch 200 or Axopatch 1C amplifier (Axon Instruments, Foster City, CA). Myocytes were allowed to settle on a poly-L-lysine coated coverslip placed on the bottom of a tissue chamber, which was mounted on the stage of a Nikon diaphot microscope. The cells were continuously superfused with a modified Tyrode’s solution of the following composition (in mM): NaCl 146, KCl 4, MgCl2 2.5, HEPES 5, dextrose 5.5, CdCl2 0.3, and MnCl2 2 (pH 7.3 with NaOH). The pipette solution had the following composition (mM): KCl 135, EGTA 10, HEPES 10, dextrose 5, ATP (Mg salt) 3, GTP (Tris salt) 0.5, pH 7.2 with KOH. The recordings were conducted at room temperature (24–26°C).

The pipette tip resistance was 0.4–1 M{Omega} when filled with the pipette solution and placed in the Tyrode’s solution. The pipette current was zeroed before forming a tight seal (>1 G{Omega}) between the pipette tip and the cell membrane. The liquid junction potential between the pipette solution and the bath solution was estimated to be about 10 mV (pipette side negative). This was corrected during data analysis. After forming the whole-cell recording configuration, a capacitive transient induced by a –10 mV step from a holding voltage of 0 mV was recorded and used for the calculation of cell capacitance. Afterwards, the series resistance (Rs) was compensated electronically by 70–90%. In our experiments, voltage error due to uncompensated Rs was estimated to be 2.4–6 mV and was not corrected.

When using 4AP, it was added from a stock solution (100 mM in water) to reach a desired final concentration of 6 mM and the pH was readjusted with HCl. TEA (20 mM) solution was made by replacing equimolar NaCl with TEA Cl.

2.4 Data acquisition and analysis
Voltage 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 2 kHz with a Bessel filter (Frequency Devices, Harverhill, MA) and digitized at a sampling interval of 0.5 or 1 ms. The voltage clamp protocols and methods of data analysis will be described in the figure legends. Clampfit of pClamp (version 6.04) was used for leak subtraction, amplitude measurement and curve fitting of time courses. PeakFit (Jandel Scientific) was used for fitting data with Boltzmann or other user-defined functions.

2.5 Membrane preparation
The rat cardiac cell membrane was prepared as described previously [23]. Briefly, the infarct zone and adjacent region from the left ventricle and the free wall of right ventricle, or the corresponding regions from control and sham-operated hearts, were dissected free and frozen at –80°C until use. All procedures of making the cell membrane preparation were performed on ice or at 4°C, and all solutions contained the following protease inhibitors: 1 mM iodoacetamide, 1 mM 1,10-phenanthroline, 2 µg/ml aprotinin, 1 mM benzamidine, 1 µg/ml pepstatin, and 0.5 mM pefebloc. Tissues were minced and homogenized in 10 volume Tris-EDTA (TE) buffer (pH 7.4), and centrifuged at 1000xg for 10 min to pellet nuclei and debris. The supernatant was centrifuged again. The supernatants from the two spins were combined and centrifuged at 40 000xg for 10 min to pellet the membrane. The pellet was resuspended in 2 ml TE containing 0.6 M KI and incubated for 10 min to disrupt the cytoskeleton. This was centrifuged at 40 000xg for 10 min. The pellet was resuspended in 2 ml TE buffer and centrifuged again at 40 000xg for 10 min. The final pellet was solubilized by incubation in 400 µl TE containing 2% Triton X-100 for 1 h. The suspension was centrifuged at 13 000xg for 10 min to remove insoluble materials. The protein concentrations of all the solubilized membrane preparations were determined using a Bicinchoninic Acid (BCA) assay kit (Pierce) and bovine serum albumin as a standard. Solubilized membranes were stored at –80°C until use.

2.6 Antibody
A rabbit anti-Kv4 polyclonal antibody was raised against a peptide of the following sequence: CLEKTTNHEFVDEQVFEES (corresponding to amino acids 484–502 in the C-terminus of Kv4.2 [26]). This sequence is highly homologous to the corresponding region of the short-isoform of Kv4.3 (different by 4 residues shown as italic above) [26]. The Kv4 antibody we made recognized two bands of approximately 72 and 77 kDa in rat heart membrane. Preincubating the Kv4 polyclonal antibody with in vitro translated Kv4.2 (70.5 kD) or Kv4.3 (short isoform, 71.4 kDa) protein diminished the intensity of both bands. Therefore, both Kv4 isoforms contributed to the two bands shown on the Western blots. Recent data suggest that in rat ventricle there is a ‘long’ isoform of Kv4.3 (73.5 kDa), that has a 19 amino acids insertion between the two underlined residues in the sequence shown above. [27] Whether this Kv4 isoform contributed to the band of a higher molecular mass on the Western blots is not clear.

2.7 Western blots
The Kv4 antiserum was purified with a protein A column (Pierce), and used at 1:1000 dilution. Aliquots (80 µg) of solubilized membranes along with prestained protein size markers (Gibco) were boiled in SDS sample buffer for 5 min, and fractionated on 7.5% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to PVDF membranes electrophoretically at 30 V and 4°C for 3 h. The membrane was blocked by incubating in Tris-buffered saline (TBS) containing 5% nonfat dry milk, 0.1% Tween 20, and 0.02% sodium azide at room temperature for >1 h. Afterwards, the membrane was incubated at 4°C overnight in a primary antibody solution prepared in TBS containing 5% nonfat dry milk, 0.2% Triton X-100 and 0.1% sodium azide at 1:1000 dilution as indicated above. After three washes using a washing solution, immunoreactive bands were visualized using the ECL kit (Amersham) following manufacturer’s instructions. Band intensities on the X-ray film were quantified by densitometry using ImagQuant software (Molecular Dynamics).

2.8 Statistical analysis
Data are presented as mean±SE. Statistical analysis was performed using SigmaStat (version 1, Jandel Scientific). For comparisons of multiple groups, one-way ANOVA was used to determine whether there were significant differences among groups. Unpaired t-test was used to determine the significance of difference between two groups. A ‘p’ value of less than 0.05 was considered to be significant. Symbols used in Figs. 3, 5, 6, 9 and 10GoGoGoGo are the following: comparison of data from the same anatomical origin between sham-operated and control hearts: +p<0.05, ++p<0.01; comparison of data from the same anatomical origin between infarcted and sham-operated hearts: *p <0.05, **p<0.01, ***p<0.001; comparison between epicardium and endocardium: #p<0.05.


Figure 3
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  		Fig. 3 (A). Representative Ito current traces recorded from myocytes isolated from right ventricle (RV, upper row), left ventricular epicardium (LV/Epi, middle row) and left ventricular endocardium (LV/Endo, lower row) of control (left column), sham-operated (middle column) and infarcted (right column) hearts. Ito was isolated from other currents as described in Fig. 2. Shown are ‘isolated’ Ito traces recorded at Vt–40 to +60 mV in 10 mV steps. Current amplitudes have been normalized by cell capacitance (pA/pF, right ordinates). (B) Summary data of peak current-voltage relationships of Ito recorded from myocytes isolated from RV, LV/Epi, and LV/Endo of control, sham-operated and infarcted hearts. Shown are mean (symbols) and SE bars, with numbers of measurements in parentheses. +p<0.01, sham vs. control. *p<0.05, **p<0.001, infarct vs. sham.

 

Figure 5
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  		Fig. 5 Background current-voltage relationships in rat ventricular myocytes isolated from right ventricle (RV, left panel), left ventricular epicardium (LV/Epi, middle panel), and left ventricular endocardium (LV/Endo, right panel) of control, sham-operated and infarcted hearts. The current-voltage relationships were constructed using the following voltage clamp protocol: from Vh–40 mV, a series of 500 ms test pulses to Vt–10 to –130 mV in 10 mV increments were applied at an interval of 5 s. Current amplitudes were measured at the end of these pulses, normalized by cell capacitance, and plotted against Vt. Shown are means (symbols) and SE bars, with numbers of measurements in parentheses. *p<0.01, infarct vs. sham.

 

Figure 6
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  		Fig. 6 K current densities and cell capacitance of rat ventricular myocytes from control (white bars), sham-operated (sham, gray bars) and infarcted (infarct, black bars) hearts three days after surgery. Tissue origins are marked on top. Peak amplitude of Ito was measured at +40 mV as shown in Fig. 2. Peak amplitude of IK was measured at +30 mV, as shown in Fig. 4. IK1 was measured at –120 mV from the background I-V relationship as shown in Fig. 5. Cell capacitance was estimated by integrating the capacitive transient induced by 10 ms voltage pulse from 0 to –10 mV and dividing the value by the voltage step. Shown are means and standard errors, with numbers of measurements marked. Statistic comparison was made between sham and control, and between infarct and sham.

 

Figure 9
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  		Fig. 9 Effects of myocardial infarction on the kinetics of Ito recovery from inactivation in left ventricular epicardial myocytes. (A) and (B) Left panels: Representative current traces recorded from a control myocyte (CLV, A) or a myocyte from an infarcted heart (ILV, B) using a double pulse protocol: from Vh–80 mV two test pulses each to +30 mV for 250 ms were applied with varying interpulse intervals. The double pulses were applied once every 10 s. Right panels: Time courses of Ito recovery from inactivation in the same myocytes. For each cell, the Ito amplitudes during the first and the second test pulses were measured as the differences between the peak outward currents and the current levels at the end of the pulses. The ratio of Ito amplitude during the second pulse to that during the first pulse was used to estimate the percentage of channels recovered from inactivation during the interpulse interval (% recovered). This time course was fit with a single exponential function. Shown are data points superimposed on curves calculated from the single exponential function with the following time constants: CLV, 26.7 ms; ILV, 89.5 ms. (C) Summary data of {tau} of Ito recovery from inactivation ({tau}restitution) in myocytes isolated from left ventricular epicardium of control, sham-operated and infarcted hearts. Numbers in bars denote those of measurements. **p<0.01, infarct vs sham.

 

Figure 10
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  		Fig. 10 Western blot analysis of Kv4 protein level in left ventricles (LV) and right ventricles (RV) of control (C), sham-operated (S) and infarcted (I) hearts. (A) Data from a representative membrane probed with a polyclonal antibody against Kv4 (for details, see Materials and methods). (B) Summary data of densitometer readings of Kv4 band intensities in LV and RV of control, sham-operated and infarcted hearts. Data were averaged from 4 experiments. For each experiment, the band intensities were normalized by that of sample from control RV (ratio to CRV). *p<0.01 infarct vs. sham.

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Characteristic of rat hearts after coronary artery occlusion
Three days after occluding the left major coronary artery, the infarct zone was readily visible in the anteroapical region of the left ventricle and extended to the anterolateral free wall to different degrees. When tissue sections were examined under the microscope, the infarct zone appeared as a transmural hypereosinophilic area with loss of cellular striations and nuclei (central zone). It was bordered by lateral ‘border zone’ in which necrotic and viable myocytes were present together with inflammatory infiltrates. The infarct (central zone) and immediately adjacent region (border zone) were used for myocyte isolation. The cells we studied were Ca-tolerant and showed clear striations but altered electrophysiological properties. Most likely they were derived from the border zone that had been affected by the infarction process. The mean cell capacitance of left ventricular myocytes isolated from the infarcted region (198±8 pF, n=37) was not significantly different from that of myocytes from sham-operated (192±6 pF, n=47) or control (193±8 pF, n=28) hearts, indicating that no overt hypertrophy developed at this stage of myocardial infarction.

3.2 Separation and quantification of depolarization-activated K currents in rat ventricular myocytes
Under our recording conditions, the Na+ current was suppressed by 0.3 mM Cd2+ in the bath solution [28]. Ca2+ currents were suppressed by removing Ca2+ from the bath solution and by adding 2 mM Mn2+. Removing extracellular Ca2+, blocking Ca2+ channels and including 10 mM EGTA in the pipette solution eliminated any role of Ca2+i-dependent currents in our measurements. In rat ventricular myocytes, at least two components of Ca2+i-independent, depolarization-activated K+ currents have been identified previously [11]. Fig. 1 shows that these two components could be readily distinguished by the differences in their voltage-dependence of inactivation and in sensitivity to blockers. We will use the same terms as used by others before [11]: ‘delayed rectifier current’ (IK) denotes the slowly decaying component that has a half-maximum inactivation voltage (V0.5) between –90 and –100 mV and is sensitive to TEA, and ‘transient outward current’ (Ito) denotes the fast decaying component that has a V0.5 around –40 mV and is sensitive to 4AP. Note that the ‘IK’ in rat ventricular myocytes is different from the rapid (IKr) or slow (IKs) delayed rectifier current in ventricular myocytes of other mammalian species in both biophysical properties and in molecular basis [23,29,30]. In the literature, different methods have been used to separate and quantify these two K+ current components in rat ventricular myocytes [11,31,32]. The method we used is illustrated in Figs. 1 and 2Go. IK was isolated from Ito by subtracting the current recorded after a Vc to –70 mV (IK inactivated while Ito fully available) from that after a Vc to –130 mV (both IK and Ito available) (Fig. 1c). Ito was separated from other currents by subtracting currents recorded at a Vh of –10 mV (when both IK and Ito were fully inactivated) from the total currents recorded at Vh–80 mV (Ito available but IK inactivated) (Fig. 2).


Figure 1
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  		Fig. 1 Separation of depolarization-activated K currents in rat ventricular myocytes into a transient outward component (Ito) and a delayed rectifier component (IK) using pharmacological agents and voltage clamp protocol. (A) and (C) 4-AP selectively blocked Ito that inactivated in a voltage range from –60 to –20 mV. (A) shows currents recorded from one cell before (control) and after the addition of 6 mM 4-AP. Inset: Voltage clamp protocol used to test the voltage-dependence of channel inactivation. From a holding voltage of –80 mV, a series of 2 s conditioning pulses to different voltages (Vc, from –130 to –10 mV in 10 mV increments) were applied at an interval of 10 s. The conditioning pulses were followed by a brief (4 ms) repolarization step to –80 mV, and then a 500 ms test pulse to +30 mV to test the availability of channels for activation. Shown are current traces recorded during the test pulses, with leak subtracted using the test pulse current following Vc to 0 mV. (C) shows peak current amplitudes during test pulses plotted against Vc before and after 4-AP. Data were from the same experiment as shown in (A). The data were fit with a double Boltzmann function:

Figure 1(1)
where Imax is the maximal peak current amplitude after Vc to –130 mV, A1 is the amplitude of current component inactivated in the more negative voltage range, V1 and k1 are the half-maximum inactivation voltage and slope factor of this component. V2 and k2 are the corresponding parameters for the current component inactivated in the less negative voltage range. (B) and (D) TEA selectively blocked IK that inactivated in a voltage range from –130 to –70 mV. (B) shows currents recorded from a cell before and after the addition of 20 mM TEA. The voltage clamp protocol was the same as that described for (A), and current traces shown were recorded during the test pulses and leak subtracted. (D) shows the double Boltzmann fit to data in (B). The IK was separated from Ito by subtracting the test pulse current following Vc to –70 mV (IK totally inactivated while Ito fully available) from that following Vc to –130 mV (both IK and Ito available), as marked in (C).

 

Figure 2
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  		Fig. 2 Quantification of Ito in rat ventricular myocytes. The left and middle panels show current traces recorded from the same cell using the voltage clamp protocols depicted in the insets. From a holding voltage of –80 mV (left panel) or –10 mV (middle panel), a 10 ms prepulse to –30 mV was followed by a 500 ms test pulse to Vt (ranging from –40 to +60 mV in 10 mV increments) applied once every 10 s. The –30 mV prepulse served to inactivate Na channels not blocked by 0.3 mM Cd in the bath solution, and thus to eliminate the interference of Na current in the measurement of Ito. The right panel illustrates difference currents (a–b) obtained by subtracting currents recorded at Vh–10 mV (b) from those recorded at Vh–80 mV (a). The Ito was measured from the peak amplitudes of the difference currents (marked by the arrow). The current amplitudes have been normalized by cell capacitance (pA/pF).

 
In almost all of the rat ventricular myocytes examined, we also observed a non-inactivating outward current at depolarized voltages. This is clearly seen in the middle panel of Fig. 2: sizable outward currents remained at Vh–10 mV when both IK and Ito were inactivated. The ionic basis for this outward current is not clear, although it was not abolished when K+ in the pipette solution was replaced by Cs+ (data not shown). Note that this outward current component was eliminated in the measurements of both IK and Ito.

3.3 K current densities in normal heart and effects of sham operation or coronary artery occlusion
In this section, data for individual current components are shown in Figs. 3–5GoGo. Fig. 6 summarizes the electrophysiological data along with measurements of cell capacitance. This figure also serves to compare the magnitudes of changes among current components.


Figure 4
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  		Fig. 4 Representative IK current traces recorded from myocytes isolated from right ventricle (RV, upper row), left ventricular epicardium (LV/Epi, middle row), and left ventricular endocardium (LV/Endo, lower row) of control (left column), sham-operated (middle column) and infarcted (right column) hearts. IK was separated from Ito as described in Fig. 1.

 
3.3.1 Transient outward current, Ito
Representative current traces of ‘isolated’ Ito recorded from myocytes of different origins are shown in Fig. 3A. The peak current density–voltage relationships are summarized in Fig. 3B. In the left ventricles of normal hearts, there was a prominent transmural gradient of Ito. At +40 mV, the mean peak current density of Ito measured from epicardial myocytes was 2.8 fold of that recorded from endocardial myocytes (Fig. 6). This is similar to the transmural gradient of Ito in rat left ventricle described by others previously [20,33]. The current density and kinetics of Ito in myocytes from the right ventricle were similar to those of Ito in epicardial myocytes from the left ventricle. This result appears to be different from that of Di Diego et al. [14] who observed a higher Ito current density in the epicardial myocytes from right ventricle than that from left ventricle in dog heart. However, a direct comparison between these two studies is difficult because we did not specifically isolate right ventricular myocytes from the epicardial region.

Sham operation did not alter the mean Ito current density in left ventricular myocytes from either epicardial or endocardial region. On the other hand, coronary artery occlusion caused a marked reduction in the Ito current density in both cell groups. The degree of reduction was more severe in epicardial myocytes (59% reduction vs. sham) than in endocardial myocytes (25%) (Figs. 3 and 6Go). As a result of this differential reduction of Ito current density, the transmural gradient of Ito in the left ventricle was reduced in infarcted hearts (ratio of epicardial Ito to endocardial Ito decreased from 2.8 to 1.9, Fig. 6).

In myocytes from the noninfarcted right ventricles of coronary artery ligated hearts, the Ito current density was not altered relative to Ito in myocytes from right ventricles of sham-operated hearts. This indicates that the reduction of Ito observed in the left ventricular myocytes was specifically linked to the infarction process, instead of trauma and stress due to the surgery. Interestingly, relative to Ito in myocytes from control right ventricles, Ito in right ventricular myocytes from both sham-operated and infarcted hearts showed a modest but significant increase (by 20% and 15%, respectively) (Fig. 6).

3.3.2 Delayed rectifier channel, Ik
Fig. 4 shows IK current traces isolated from Ito and the non-inactivating outward current component as described above (Fig. 1C). The current traces manifested a slow decay during the 500-ms depolarization pulses. IK amplitudes were quantified from the peaks of these current traces. Data are summarized in Fig. 6. In the control hearts, the mean IK current density in left ventricular myocytes of the epicardial origin was 21% higher than that of endocardial origin, a transmural gradient much smaller than that of Ito.

Sham operation did not affect the IK current density recorded from left ventricular myocytes of either epicardial or endocardial origin. On the other hand, myocardial infarction caused a marked reduction in the IK current densities of both cell groups. The degree of IK reduction was comparable between the two groups: 39% decrease in the epicardial myocytes and 35% decrease in the endocardial myocytes (relative to IK in myocytes from sham-operated hearts, Fig. 6). This reduction in IK current density was specifically related to the infarction process, because IK current density in right ventricular myocytes from infarcted hearts was not different from that of sham-operated hearts. Relative to the mean IK current density in control right ventricular myocytes, the IK amplitudes were modestly but significantly increased in right ventricular myocytes from both sham-operated (by 20%) and infarcted (by 14%) hearts (Fig. 6). The degree of changes was similar to the increase in Ito current density in right ventricular myocytes from sham-operated and infarcted hearts.

3.3.3 Inward rectifier channel, IK1
Fig. 5 illustrates average ‘background’ current density-voltage relationships recorded from myocytes of different origins. The I-V relationships in the voltage range negative to –40 mV are mainly determined by the inward rectifier channel, IK1 [12]. The I-V relationship of IK1 in rat ventricular myocytes does not display a prominent N-shape or a negative slope region as in ventricular myocytes from most other species [34,35]. Therefore, we used the steady-state inward current recorded at –120 mV to quantify the IK1 current density. Data are summarized in Fig. 6.

In left ventricular myocytes from control hearts, the mean IK1 current density recorded from endocardial myocytes was 24% higher than that recorded from epicardial myocytes (Fig. 6). This transmural gradient is similar to that described for IK1 in cat left ventricular myocytes [17], and is opposite to the transmural gradient of Ito and IK. The IK1 current density recorded from right ventricular myocytes was intermediate between the values recorded from the epicardial and endocardial myocytes of the left ventricle.

Sham operation did not alter the IK1 current density in myocytes from all three regions (Fig. 6). In the left ventricle, myocardial infarction caused a reduction in the IK1 density in epicardial myocytes. However, the IK1 current density was not significantly affected in endocardial myocytes. In right ventricular myocytes, myocardial infarction was associated with an increase in the IK1 current density relative to that of sham-operated or control hearts.

3.4 Effects of myocardial infarction on the voltage-dependence and kinetics of Ito gating in left ventricular epicardial myocytes
The above data show that three days after coronary artery ligation the most prominent change among the three K channels examined here was a reduction of Ito current density in left ventricular epicardial myocytes. To study whether this decrease in Ito current amplitude could be explained by alterations in the channel’s gating behavior, we examined the voltage- and time-dependence of Ito gating. Fig. 7A shows that the voltage-dependence of Ito activation was not affected by myocardial infarction. In addition, myocardial infarction did not shift the voltage-dependence of Ito inactivation (Fig. 7B). These data suggest that the reduction of Ito amplitude in infarcted hearts could not be accounted for by changes in the voltage-dependence of activation or inactivation. Fig. 7B also shows that the voltage-dependence of inactivation of IK in left ventricular epicardial myocytes was not altered by myocardial infarction.


Figure 7
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  		Fig. 7 (A) Voltage-dependence of Ito activation in left ventricular epicardial myocytes from control, sham-operated and infarcted hearts. For each cell, the peak Ito amplitudes were measured using the method described in Fig. 2. This was divided by the driving force (Vt–Vrev, where Vrev was the calculated reversal potential of Ito, Vrev=RT/F(ln[K]o/[K]i)=–90 mV) to estimate the peak chord conductance activated at each Vt. The chord conductance was normalized by that at +60 mV. The relationships between Vt and normalized chord conductance was fit with a simple Boltzmann function to estimate the half-maximum activation voltage (V0.5) and slope factor (k):

Figure 7(2)
The curves superimposed on the data points were calculated from Eq. (2) with best-fit parameter values. (B) Voltage-dependence of IK and Ito inactivation. The voltage clamp protocol was as described for Fig. 1. For each cell, the IK inactivation curve was constructed by subtracting the peak amplitude of test pulse current recorded following Vc to–70 mV from those recorded following Vc to –130 to –80 mV. Data were fit with Eq. (2). The curves superimposed on the data points were calculated from Eq. (2) with the following mean parameter values (in mV): control, V0.5=–100±1, k=–9.6±0.3; sham-operated, V0.5=–98±1, k=–9.7± 0.4; infarct, V0.5=–93±1, k=–7.4±0.4. The Ito inactivation curves were constructed by normalizing the test pulse current amplitudes following Vc to –60 to –20 mV to that following Vc to –70 mV. The curves superimposed on the data points were calculated from Eq. (2) with the following mean parameter values (in mV): control, V0.5=–40±1, k=–4.0±0.1; sham-operated, V0.5=–38–1, k=–3.9±0.1; infarct, V0.5=–40±1, k=–4.0±0.1. For all three panels, symbols represent mean values and SE bars are smaller than symbols. Numbers in parentheses denote numbers of measurements.

 
Although the voltage-dependence of Ito inactivation was not altered in infarcted hearts, there were modest but significant changes in the kinetics of Ito inactivation and recovery from inactivation. As shown in Fig. 8A, the time course of Ito inactivation during the initial 200 ms of depolarization could be well described by a single exponential function. The average time constant of inactivation ({tau}decay) of Ito recorded at four different test voltages from different cell groups are summarized in Fig. 8B. At all four voltages, the {tau}decay values were significantly prolonged in myocytes from infarcted hearts, but not significantly altered in sham-operated hearts. The time course of Ito recovery from inactivation ({tau}restitution) could be well described by a single exponential function. Examples from a control myocyte and a myocyte from an infarcted heart are shown in Fig. 9A and B, respectively. Fig. 9C presents the summary data of {tau}restitution recorded from different cell groups. The mean time constant of Ito recovery from inactivation was not affected by sham operation, but was prolonged in myocytes from infarcted hearts.


Figure 8
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  		Fig. 8 Effects of myocardial infarction on the kinetics of Ito decay during depolarization. (A) Ito decay followed a single exponential function. Shown are representative Ito current traces recorded from a control left ventricular epicardial myocyte at four different test voltages (0 to +30 mV in 10 mV increments). The superimposed curves are single exponential fit to the current traces between peaks and 200 ms, with time constants ({tau}, in ms) marked. (B) Summary data of {tau}Decay at various Vt recorded from myocytes isolated from left ventricular epicardium of control, sham-operated and infarcted hearts. Shown are means (symbols) and SE bars, with numbers of measurements in parentheses. **p<0.01, ***p<0.001, infarct vs. sham.

 
3.5 Immunoblot analysis of Kv4 subunits in control, sham-operated and infarcted hearts
Our electrophysiological experiments revealed that the Ito amplitude was reduced in left ventricular myocytes from infarcted hearts relative to control or sham-operated hearts. On the other hand, the current amplitude was increased in right ventricular myocytes from infarcted and sham-operated hearts relative to control hearts. To test whether there were corresponding changes in the protein level of the major subunits for Ito channels in rat ventricles (Kv4.2 and Kv4.3) [22–24], we performed Western blot analysis using a polyclonal antibody that could recognize both Kv4.2 and Kv4.3 (see Materials and methods). To facilitate quantitative comparisons, each Western blot compared the protein levels in membrane preparations from left and right ventricles of three animals: control, sham-operated and coronary artery-ligated. To combine data from different Western blots, samples from control right ventricular myocardium (CRV) were used as an external control, i.e. the densitometer readings were normalized by the CRV band intensity in the same blot (ratio to CRV).

Fig. 10A shows data from a representative experiment. Summary data are presented in Fig. 10B. The only statistically significant change induced by myocardial infarction was a reduction in the Kv4 protein level (Kv4.2+Kv4.3) in left ventricular myocardium from infarcted hearts relative to that from sham-operated hearts. The Kv4 protein level in right ventricular myocardium from infarcted hearts tended to be higher than that in sham-operated hearts. Furthermore, the signals in both left and right ventricles of sham-operated hearts appeared to be lower than the corresponding signals in control hearts. However, these latter differences were not statistically significant.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
4.1 Major findings of the present study
Our data show that the three types of K channels we examined here, Ito, IK and IK1, had different patterns of distribution in normal rat ventricles. Sham operation did not affect the mean current density of any of these three K channels in the left ventricle, whereas coronary artery occlusion caused a reduction in all three. The mean current density of IK was reduced by a similar amount between the epicardium and endocardium. However, the degree of reduction was not homogeneous for Ito or IK1. The mean Ito current density was reduced more in the epicardial than in the endocardial myocytes. IK1 was reduced in epicardial, but not in endocardial, myocytes.

It is important to point out that infarction-induced changes in cellular function are inhomogeneous in the border zone myocardium. The most severely damaged myocytes probably died during the disaggregation procedure. Among the myocytes that survived the disaggregation procedure, we sampled from those showing clear striations. Therefore, it is possible that the changes in current densities and Ito gating kinetics we reported here represent the lower end of abnormalities induced by the subacute phase of myocardial infarction in rat heart.

In control hearts, the mean cell capacitance of right ventricular myocytes was smaller than that of left ventricular myocytes (Fig. 6). In infarcted hearts, the mean cell capacitance of right ventricular myocytes was increased relative to that in the normal heart, along with an increase in Ito and IK current densities. Since similar changes also occurred in the right ventricles of sham-operated hearts, they were not specifically associated with the infarction process, but might be related to trauma or stress induced by the surgery. However, neither the increase in cell capacitance nor the increase in Ito and IK current densities was observed in left ventricular myocytes from sham-operated hearts, suggesting that these nonspecific changes were not homogeneous between the two ventricles. Although a reduction of Ito current density has been described for a number of rat models of myocardial hypertrophy [36,37], our observations reported here (i.e. increase in cell capacitance accompanied by a higher Ito current density in right ventricular myocytes of sham-operated and coronary artery-ligated hearts) indicate that cellular hypertrophy is not always linked to a reduction in Ito.

4.2 Alterations in Ito channel function in rat heart with subacute myocardial infarction
Since we controlled the intra- and extra-cellular ionic composition in our electrophysiological experiments, the changes in membrane currents we observed were probably not related to alterations in cellular milieu associated with acute and subacute myocardial infarction (e.g., changes in [ATP]i, [K+]o, [K+]i, [Na+]i, [Ca2+]i, pHi or pHo [1]). Instead, they were likely due to alterations in protein expression, channel subunit composition, or post-translational modification. We performed Western blot analysis to see whether there were changes in the protein level of Ito channel subunits, and whether these changes could be correlated with the alterations in the Ito current amplitude. In rat ventricles, Ito channels are formed by two major {alpha} (pore-forming) subunits: Kv4.2 and Kv4.3 [22–24]. Both isoforms could be recognized by our antibody. We saw a marked reduction in the Kv4 protein level in left ventricular myocardium of infarcted hearts relative to that of control or sham-operated hearts. This was probably due to a reduction of both Kv4.2 and Kv4.3, because a recent preliminary report showed that the mRNA levels of both isoforms were reduced in rat ventricles three-days after occluding the major left coronary artery [38]. However, a direct quantitative comparison between the reduction in Ito current density and changes in Kv4 protein level is difficult because Ito showed a differential reduction in epicardial and endocardial myocytes while the Kv4 protein was measured from both cell populations.

The reduction in Kv4 protein level is consistent with our expectation. However, a simple reduction of Kv4 protein expression can not account for all the observed changes in Ito. First, a decrease in the Kv4 protein level can not explain why the apparent kinetics of Ito were altered in left ventricular myocytes from infarcted hearts. Second, in right ventricles of sham-operated hearts there was a dissociation between the change in Ito current density (increase) and the change in Kv4 protein level (decrease although not statistically significant). Therefore, it is likely that other change(s) also contributed to the alterations in Ito reported here. One possibility is that in rat ventricular myocytes Kv4.2 and/or Kv4.3 is associated with another subunit that can regulate its expression in the cell membrane and modulate its kinetics of inactivation and recovery from inactivation. It has been shown that coexpression of small molecular weight (2–4 kb) poly(A)+ RNA from rat brain can increase the cell membrane expression and accelerate the rate of inactivation and recovery from inactivation of mouse Kv4.1, as well as Kv4.2 and Kv4.3 [26,39,40]. A decrease in the expression of this unidentified subunit in infarcted left ventricles may reduce Ito current density and slow the kinetics of inactivation and recovery from inactivation. An increase in the expression of this subunit in right ventricles of sham-operated hearts might cause a greater Ito current density, even though the Kv4 protein level is not altered or even reduced.

4.3 Comparison with previous studies
Our results are distinctly different from findings in two previous reports examining the effects of myocardial infarction on K channel function and expression in rat ventricle 3–4 weeks after coronary artery ligation [32,41]. We studied a much earlier stage of myocardial infarction (3 days vs. 3–4 weeks post coronary artery occlusion). At this early stage, there was no overt cellular hypertrophy in the infarcted ventricle, while cellular hypertrophy was prominent in the more chronic model [32]. The densities of Ito and IK were reduced in myocytes from infarcted ventricle both in our study and in the previous report (termed Ito–f and Ito–s, respectively [32]). However, the underlying mechanisms might be different. During the subacute phase of myocardial infarction studied here, the decrease in Ito current density was accompanied by a modest but significant slowing of inactivation and recovery from inactivation. This suggests that not only was there a reduction of channel expression, but also an alteration in the channel protein or subunit composition. During the chronic phase of myocardial infarction, Ito current density was reduced without any detectable changes in the kinetics.

The changes we observed were similar to the reported changes in K channel function in the epicardial border zone from canine hearts 5 days after coronary artery occlusion [4]. In canine ventricular myocytes isolated from the epicardial border zone, Ito current density is reduced along with a modest slowing of inactivation and recovery from inactivation. There also was a decrease in the IK1 current density. However, more experiments are needed to test whether the similarity extends to changes in K channel protein expression.

Time for primary review 26 days.


   Acknowledgements
 
The authors would like to thank Drs. B. F. Hoffman, and P. A. Boyden for helpful discussion of this work, and Pat McLaughlin for making rat ventricular myocytes. This work was supported by HL-30557 and HL-46451 from National Heart, Lung, and Blood Institute, National Institutes for Health, Bethesda, MD, and a grant-in-aid from American Heart Association/New York City Affiliate.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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