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J Am Soc Nephrol 11:1589-1599, 2000
© 2000 American Society of Nephrology

An Altered Repolarizing Potassium Current in Rat Cardiac Myocytes after Subtotal Nephrectomy

PAUL DONOHOE*, BRUCE M. HENDRY*, OMAL V. WALGAMA{ddagger}, FEDERICA BERTASO*, DEBORAH J. HOPSTER{dagger}, MICHAEL J. SHATTOCK{ddagger} and ANDREW F. JAMES*

* Department of Renal Medicine, Guy's King's and St. Thomas' School of Medicine, King's College London, United Kingdom.
{dagger} Department of Pathology, Guy's King's and St. Thomas' School of Medicine, King's College London, United Kingdom.
{ddagger} Rayne Institute, St. Thomas' Hospital, Guy's King's and St. Thomas' School of Biomedical Sciences, King's College London, United Kingdom.

Correspondence to Dr. Andrew F. James, Department of Renal Medicine, GKT School of Medicine, King's College London, Bessemer Road, London SE5 9PJ, United Kingdom. Phone: +44 (0)20 7928 9292, ext. 3372; Fax: +44 (0)20 7928 0658; E-mail: andrew.f.james{at}kcl.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. Renal failure in humans is associated with electrocardiographic changes including altered QT interval dispersion, which suggests that cardiac myocyte repolarization is abnormal and which appears to correlate with cardiac prognosis. In this study, cardiac myocyte repolarizing currents have been studied in isolated cells from rats 8 wk after subtotal nephrectomy (SNx), using sham-operated animals as controls. In addition, monophasic cardiac action potentials were recorded from the epicardial surface of the left ventricle (LV) apex, LV base, and the right ventricle of isolated perfused hearts paced at 320/min. SNx was associated with cardiac hypertrophy and histologic evidence of myocardial fibrosis, but SNx rats were not hypertensive. Repolarizing K+ currents were measured using whole-cell patch-clamp, and 4-aminopyridine (4-AP)-sensitive transient outward (Ito) and 4-AP-insensitive sustained outward (Iso) components were quantified. After SNx, Ito was increased by two to threefold at voltages from -30 to +60 mV and showed increased heterogeneity. For example, at 0 mV voltage clamp pulse, the median Ito was increased from 3.23 pA/pF in control myocytes (interquartile range 3.20 pA/pF, n = 24) to 5.86 pA/pF in SNx myocytes (interquartile range 7.32 pA/pF, n = 21, P < 0.005). The kinetics of inactivation of Ito were altered after SNx with slowing both of the onset and the recovery from inactivation. The mean time constant of inactivation at +30 mV after SNx was 14.2 ± 1.6 ms (n = 20) compared with control values of 9.8 ± 0.6 ms (n = 23, P < 0.05). Neither Iso nor inward rectifier K+ currents were altered after SNx. The action potential duration (APD50) at the left ventricular base was approximately 20% shorter (P < 0.02) in hearts from SNx rats compared with controls. 4-AP (2 mM) prolonged the APD50 in all regions in hearts from both SNx and control rats and abolished the APD50 shortening in SNx. These results indicate that abnormalities of the cardiac transient outward K+ current contribute to alterations in the cardiac action potential in renal failure and warrant further investigation because they may contribute to altered repolarization and arrythmogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic renal failure is associated with a greatly increased risk of cardiac death (1). Similar to acceleration of coronary atheroma, end-stage renal failure is associated with a cardiomyopathy characterized by left ventricular hypertrophy (LVH), globally reduced cardiac output, systolic and diastolic dysfunction, and intercardiomyocytic fibrosis (2,3,4,5,6). In addition to contractile dysfunction in renal failure, there is a proarrhythmogenic tendency, with increased occurrence of ventricular premature beats, couplets, runs of ventricular tachycardia, and late ventricular depolarizations (7,8,9). Increased electrocardiogram QT interval dispersion has also been described (10). The origins of this increased QT dispersion are poorly understood, but are likely to represent regional abnormalities of myocardial repolarization (11).

It has been the subject of debate whether a specific "uremic cardiomyopathy" is present or whether the cardiac dysfunction is a result of a combination of hypertension, chronic fluid overload, anemia, systemic acidosis, and hyperparathyroidism (12,13). The subtotal nephrectomy (SNx) model in the rat in which on kidney and two-thirds of the remaining kidney are removed provides a means of investigating the basis of this cardiomyopathy. The hearts of SNx rats develop LVH and intercardiomyocytic fibrosis, and the isolated perfused hearts show a reduced cardiac output when compared with shamoperated controls (14,15,16). Electrically paced isolated single ventricular myocytes show contractile abnormalities, demonstrating that the cardiomyopathy occurs at the single cell level (17). Alterations in Ca2+ handling have also been reported in isolated myocytes from SNx hearts (18,19). We have previously reported an increase in the rate of inactivation of the L-type Ca2+ current in isolated cardiac myocytes from SNx rats (20).

Voltage-gated K+ currents play an important role in the modulation of cardiac excitability and conduction and in the repolarization of the cardiac action potential, K+ efflux returning the membrane voltage to the resting membrane potential. The 4-aminopyridine-sensitive transient outward current (Ito) is a K+ current that activates rapidly on depolarization and subsequently inactivates and is found in cardiac ventricular myocytes from many species, including rat and human (21,22,23,24,25). It is thought to contribute to early phase repolarization and play an important role in modulating cardiac excitability and conduction (26). A 4-aminopyridine-insensitive sustained outward current (Iso) remains after inactivation of Ito (22,25). The properties of Ito have been shown to be altered in ventricular myocytes from a number of rat models of myocardial hypertrophy (27,28,29,30,31) and from patients with terminal heart failure (24). It has been suggested that abnormalities in Ito may lead to cardiac arrhythmias (26). In this study, we have examined the voltage-gated K+ currents in ventricular myocytes isolated from the rat subtotal nephrectomy model of renal failure compared with sham-operated controls using whole-cell patch-clamp techniques. In addition, we have recorded monophasic action potentials (MAP) from the epicardial surface of paced, isolated perfused hearts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Model
Six week-old 200-g male Wistar rats were given renal failure by two-stage SNx under general anesthesia, as described previously (14). Sham-operated control rats underwent two-stage bilateral renal decapsulation. After surgery, animals were pair-fed standard rat chow and housed in individual cages, with free access to water. Systolic BP was measured by tail-cuff sphygmomanometry at 4 and 8 wk after the second surgical procedure (Harvard Instruments) following acclimatization in restraining cages. Rats were placed in metabolic cages the day before sacrifice, and urine samples were obtained for creatinine clearance estimation. Eight weeks after the second surgical procedure, animals were sacrificed for myocyte isolation. At the time of sacrifice, blood samples were taken from the thoracic cavity and subsequently analyzed for urea, creatinine, hemoglobin, hematocrit, pH, and HCO3. No animals were excluded on the basis of these blood samples. All procedures were performed according to UK Government guidelines and legislation.

Histology
A total of five hearts from control rats and six hearts from SNx rats were examined in a blinded manner 8 wk after the second surgical stage, by one histopathologist. Whole hearts, left ventricles (including septum), and right ventricles were weighted. Sections of left ventricle were stained with hematoxylin and eosin and examined histologically.

Myocyte Isolation
Ventricular myocytes were isolated by retrograde perfusion of the heart via the aorta with an enzyme-containing solution as described previously (17). Briefly, hearts were excised quickly under deep general anesthesia into carboxygenated ice-cold bicarbonate-buffered solution (pH 7.4) and mounted on a modified Langendorff apparatus. Hearts were perfused at 37°C for 5 min followed by a solution containing low Ca2+, for at least 5 min. Finally, hearts were perfused with low Ca2+ solution containing collagenase class 2 (184 U/ml; Worthington Biochemical, Freehold, NJ) for 8 to 10 min. Left ventricular tissue fragments were finely minced, and the suspension was washed and resuspended in low Ca2+ solution. Viability ranged from 20 to 50% viable myocytes, defined as Ca2+-tolerant, single, rodshaped striated cells. Cells were stored in the resuspension solution at room temperature and used in patch-clamp experiments within 6 h of isolation.

Electrophysiologic Experiments
Isolated cells were perfused with experimental solution (in mM): 150 NaCl, 5.4 KCl, 2 MgCl2, 10 Hepes, 10 glucose, 0.5 CaCl2, pH 7.4, with 1 M NaOH) at 37°C at a flow rate of 2 to 3 ml/min. Patch pipettes had a tip resistance of 2 to 4 M{Omega} when filled with the K+-rich pipette solution (in mM): 130 KOH, 130 L-aspartic acid, 4 NaCl, 5 CaCl2, 10 ethylene glycol-bis(ß-aminoethylether)-N,N,N',N'-tetra-acetic acid (EGTA) (free [Ca2+] = 72 nM, corresponding to a pCa of 7.14), 10 Hepes, 4 Mg2+-ATP, and 0.1 Na+2-GTP (pH 7.4 with KOH) (32). All reagents were obtained from Sigma (United Kingdom).

Whole-cell patch-clamp recordings were made using EPC-9 (HEKA, Germany) and Axopatch 200B (Axon Instruments) amplifiers. Data were saved to the hard disk of an Apple computer using Pulse/PulseFit software (HEKA). Series resistance and capacitance compensation were applied electronically. For current-voltage relation experiments, a family of 19 voltage pulses of 200 ms duration increasing in 10-mV increments from -100 to +60 mV were applied at 4-s intervals from a holding potential of -80 mV. Before each voltage-pulse, a 30-ms prepulse to -40 mV was applied to inactivate the Na+ current. Verapamil (10 µM) was added to the bath solution to block the L-type Ca2+ current. Verapamil was found to have little effect on Ito (inhibited by 7%) but blocked Iso (inhibited by 49%) in control experiments, consistent with other reports (33). Currents were reproducible and showed minimal rundown during the course of experiments. An estimate of the voltage dependence of steady-state inactivation of K+ currents was obtained using a series of prepulses (300 ms) increasing from -80 to +20 mV in 5-mV increments followed by a 200-ms test pulse to +40 mV applied at 4-s intervals. The time course of recovery of Ito from inactivation was investigated using a series of paired voltage pulses of 200 ms to +40 mV from a holding potential of -80 mV. The time interval ({Delta}t) between the end of the first pulse and the start of the second pulse was increased in 5-ms increments from 15 to 75 ms. Pairs of pulses were applied every 3 s. Diagrams of the voltage protocols used are included as insets in the appropriate figures.

Epicardial Monophasic Action Potentials
Control and SNx rats were prepared surgically as before. A total of three control and five SNx rat hearts were used to record MAP, again at 8 wk after the second surgical procedure. Rats were anesthetized with 0.1 ml/100 g SalanaxTM intraperitoneally (Pentobarbitone, Rhone Merieux, Athens, GA) and treated with 0.15 ml of 1:5000 heparin solution (Leo Laboratories, Princes Risborough, UK). Depth of anesthesia was assessed by hind leg withdrawal reflex. Hearts were excised into ice-cold Krebs-Henseleit buffer ([in mM]: 119 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11.5 glucose, 1.2 CaCl2, pH 7.4 with 1 mM NaOH, bubbled with 95% O2-5% CO2 mix) and mounted on a Langendorff perfusion apparatus. Perfusion was commenced with Krebs-Henseleit buffer at 37°C in a retrograde manner with a perfusion pressure of 100 cm of H2O, resulting in flow rates ranging from 10 to 15 ml/min. Hearts were paced at 320 beats/min. After a 20-min stabilization period, MAP values were recorded using an epicardial suction electrode, positioned in turn at the apex and base of the left ventricle, and at the right ventricle.

Statistical Analyses
Data are expressed as mean ± SEM and statistical significance was assessed by either two-tailed t test or ANOVA (P < 0.05 was considered significant). The distribution of the current density was found to be non-Gaussian and therefore statistical significance was assessed by a two-tailed nonparametric Mann—Whitney U test, and P < 0.05 was considered significant. Curve fitting was performed by nonlinear least squares using IgorPro version 2.1 (WaveMetrics, Lake Oswego, OR).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animal Data
SNx rats (n = 13) had a fourfold increase in mean serum urea compared with control rats and a 2.7-fold increase in mean serum creatinine (Table 1). The creatinine clearance of SNx rats was reduced to 29% of control rats. There was no significant difference in 4- or 8-wk systolic tail BP, blood pH, or HCO3- between SNx and control rats. SNx rats had mild anemia and reduction in hematocrit compared with control rats (Table 1).


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  		Table 1. Data from control and SNx animalsa
 

Histology
The heart weight to body weight ratio of the SNx rats was increased by 62.5% compared with controls (P < 0.05) (Figure 1), demonstrating significant cardiac hypertrophy. Furthermore, the left ventricle (including septum) to right ventricle weight ratio (LV + S/RV) was increased by 70% in SNx hearts compared with controls (P < 0.05) (Figure 1), indicating LVH. Histologic examination showed scattered areas of fibrosis and fibroblast proliferation, predominantly in the subendocardium, in hearts from SNx but not control rats (Figure 2).



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  		Figure 1. Subtotal nephrectomy produces left ventricular hypertrophy. (A) Heart weight to body weight ratios of control (n = 5) and subtotal nephrectomy (SNx) (n = 6) rats. (B) Left ventricular (LV) (including septum) to right ventricular (RV) weight ratios. *P < 0.05.

 


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  		Figure 2. Hematoxylin and eosin-stained sections of left ventricle from control (A) and SNx (B) rats. Focal subendocardial area of fibrosis and fibroblastic proliferation in SNx rat is shown in Panel B. Magnification, x200.

 

Electrophysiologic Experiments
Voltages from -30 mV to positive activated voltage-gated outward currents in myocytes from both SNx and control rats. The currents activated rapidly to a peak (Ipk) and subsequently inactivated to a sustained level (Iso) at the end of the pulse (Figure 3, A through C). Voltages negative to -60 mV activated an inward current that represented the inwardly rectifying background K+ current (IK1). A rapidly activating and inactivating transient outward current (Ito) could be identified as the difference between the peak and sustained outward currents (Ito) (Figure 3C). The mean current—voltage relations for Ito and Iso in myocytes from both control and SNx rats are shown in Figure 3D. Mean Ito was strikingly increased by two-to threefold in SNx myocytes compared with control myocytes for all voltages from -30 mV to +60 mV. There was no difference between SNx and control myocytes in either mean Iso or IK1 (Figure 3D).



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  		Figure 3. Current—voltage relations of the 4-aminopyridine-sensitive transient outward current (Ito) and the 4-aminopyridine-insensitive sustained outward current (Iso). (A) The voltage pulse protocol. (B) A family of currents elicited using the voltage pulse protocol shown, with test pulses increasing from - 100 mV to + 60 mV in 10-mV increments. (C) An example current showing that Ito was measured as the difference Ipeak — Iso. (D and E) Current voltage relations for Ito (D, circles) and Iso (E, squares) recorded from myocytes from control rats (open symbols) and SNx rats (filled symbols). Data are shown as the mean and the error bars represent the SEM. The mean whole-cell capacitance of myocytes from SNx rats (121 ± 6 pF, n = 21) was not significantly different from control rats (116 ± 7 pF, n = 24).

 

There was considerable heterogeneity between cells in the magnitude of Ito. This is illustrated by the population histogram of Ito density for control and SNx myocytes (Figure 4). Ito recorded at 0 mV was normalized to cell capacitance as a measure of membrane surface area. SNx was associated both with a marked increase in the magnitude of Ito (control, median Ito 3.23 pA/pF, n = 24; SNx, median Ito 5.86 pA/pF, n = 21, P < 0.005) and a considerable increase in the heterogeneity of the current (control, interquartile range [iqr] 3.20 pA/pF; SNx, iqr 7.32 pA/pF). In contrast to Ito, there was very little heterogeneity between cells in the magnitude of Iso (compare Figure 3, D and E).



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  		Figure 4. Distribution of Ito (normalized to capacitance) at 0 mV in control (A) and SNx (B) myocytes. The median Ito was 3.23 pA/pF in control myocytes (interquartile [iqr] range 3.20 pA/pF, n = 24) and 5.86 pA/pF in SNx myocytes (iqr 7.32 pA/pF, n = 21).

 

The nature of Ito and Iso was investigated further. Ito was abolished and Iso was markedly reduced in experiments in which intracellular K+ ions were replaced by Cs+ ions in the pipette solution (data not shown), demonstrating that these currents were carried predominantly by K+ ions. The K+ channel blocker 4-aminopyridine (4-AP) selectively blocked Ito in a concentration-dependent manner (2 mM: 66 ± 17% block; 10 mM: 100 ± 0% block) with little effect on Iso (2 mM: 2.0 ± 3.8% block; 10 mM: 26.1 ± 9% block). SNx had no effect on the sensitivity of Ito and Iso to 4-AP (data not shown). The voltage dependence of K+ current inactivation was examined by a two-pulse protocol (Figure 5, inset). Only Ito (and not Iso) demonstrated voltage-dependent inactivation, which could be fitted by a Boltzmann equation (see Figure 5 legend). Taken together, these results demonstrate that Ito and Iso represent distinct K+ currents. The fits to the voltage-dependent inactivation of Ito in SNx and control myocytes were virtually superimposed, demonstrating that there was no significant change in the voltage dependence of inactivation of Ito in renal failure. Ito inactivated with a half-maximal voltage of -50.1 mV in control myocytes and -50.8 mV in SNx myocytes (Figure 5).



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  		Figure 5. Voltage-dependent inactivation of Ito and Iso. (A) The voltage pulse protocol. (B) Sample family of currents with voltage pulse protocol is shown. Currents were normalized to the maximal Ito or Iso current obtained after a prepulse to -75 mV. (C) Dependence of Ito (circles) and Iso (squares) on the prepulse voltage in control (open symbols) and SNx (filled symbols) myocytes. Data are the mean ± SEM. Solid lines show the fits to a Boltzmann equation, I/Imax = 1/(1 + exp((Vm - V1/2max)/Vs) + c, where Vm is the membrane potential, V1/2max is the voltage of half-maximal inactivation, and Vs is the slope factor. The fitted parameters were V1/2max = -50.1 mV (Vs = 9.4 mV) in control myocytes and V1/2max = -50.8 mV (Vs = 9.8 mV) in SNx myocytes.

 

The effect of SNx on the time course of inactivation of Ito elicited by voltages from +30 to +60 mV was examined. Currents were normalized to the peak current obtained soon after depolarization (Ipk), and a decaying exponential curve was fitted to the inactivation phase of the currents (Figure 6). In the majority of cells (30 of 45), the inactivation of Ito was adequately described by a single exponential. The time constant ({tau}, approximately 10 ms) did not show voltage dependence. In the remaining 33% of cells, two exponential components (with time constants {tau}1 and {tau}2) were necessary to describe the time course of inactivation. The slow time constant ({tau}2) had a value of about 70 ms and accounted for approximately 12% of the maximal outward current at +30 mV. The {tau}1 values for these cells were comparable with the {tau} values of the cells described by a single exponential, suggesting that these time constants described a relatively fast inactivation process common to all of the cells. Therefore, the data for the single {tau} and the {tau}1 of the double exponential fits were pooled and referred to as {tau}fast. The {tau}fast in SNx myocytes ({tau}fast 14.2 ± 1.6 ms at +30 mV, n = 20) was prolonged compared with control myocytes ({tau}fast 9.8 ± 0.6 ms at +30 mV, n = 23, P < 0.05) for all voltages from +30 mV to +60 mV (Figure 6B). There was no difference in the proportion of cells in which a slow component of inactivation could be identified between SNx rats (7 of 21, 33%) and control rats (8 of 24, 33%).



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  		Figure 6. Time course of Ito inactivation. Currents were evoked by depolarization to +30 mV and normalized to the peak outward current. Normalized currents were fitted to the equation I/Ipeak = A1 exp(-t/{tau}) + c. In a minority of cells (33%), the inactivation of the currents was only described as the sum of two exponentials (I/Ipeak = A1 exp(-t/{tau}1) + A2 exp(-t/{tau}2) + c). (A) Example of normalized SNx and control currents at +30 mV showing single exponential time course. Note the slower inactivation in SNx cells. (B) Mean ± SEM of {tau}fast of inactivation of currents for voltages between +30 and +60 mV, for both control (open symbols) and SNx (filled symbols). {tau}fast of SNx myocytes was significantly slower than control myocytes over the voltage range +30 to +60 mV (P < 0.05, ANOVA).

 

The time course of recovery from inactivation of Ito was examined using a paired-pulse protocol (Figure 7A). Ito showed a monophasic recovery from inactivation with time constant {tau}rec, but was not completely recovered at the longest interpulse intervals used (75 ms, Figure 7C). The time course of recovery of Ito was significantly slowed in myocytes from SNx hearts compared with control hearts (P < 0.05, ANOVA). The fitted value of {tau}rec was slower in SNx (20.6 ± 2.5 ms, n = 13) compared with control (12.9 ± 1.2 ms, n = 12, P < 0.01).



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  		Figure 7. Time course of recovery from inactivation of Ito. A paired-pulse protocol (A) in which the time between the two pulses was varied ({triangleup}t) was used to examine the time course of recovery from inactivation of Ito (sample family of current traces in B). (C) Dependence of Ito recorded during the second pulse on {triangleup}t for control (open circles) and SNx (filled circles) myocytes. Data are mean ± SEM of Ito normalized to the maximal current activated during the first pulse. Solid lines represent fits to the equation I/Imax = A0 + A1(1 - exp(-t/{tau}rec). (D) Individual values of {tau}rec were obtained for each cell studied, and the mean (± SEM) values of {tau}rec were 20.6 ms (± 2.5 ms) in SNx myocytes and 12.9 ms in control myocytes (± 1.2 ms, P < 0.01).

 

The duration of the action potential in isolated perfused hearts from control (n = 3) and SNx (n = 5) animals was examined by MAP recordings from the epicardial surface of the ventricle. Figure 8 shows sample recordings of MAP from the apex and the base of the left ventricle (LV) and from the right ventricle (RV). The mean action potential duration from each region of the heart, calculated as the time between the beginning of the upstroke and 50% repolarization of the peak of the action potential (APD50), is shown in Table 2. APD50 was shorter in the LV apex and RV compared with the LV base. SNx shortened the APD50 by approximately 20% in the LV base (P < 0.02). Perfusion with 2 mM 4-AP prolonged the APD50 in all regions of the ventricle of hearts from both control and SNx rats. In the LV base, the APD50 was prolonged by 13% to 30.1 ± 2.3 ms in hearts from control animals and by 38% to 27.2 ± 2.9 ms in hearts from SNx animals. Thus, perfusion with 2 mM 4-AP abolished the difference in APD50 at the LV base between hearts from control and SNx animals.



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  		Figure 8. Monophasic action potentials recorded from the epicardial surface of isolated, perfused hearts from control and SNx rats. Hearts were paced at 320 bpm. The action potential amplitude was measured as the difference between the diastolic voltage and the peak of the action potential. Dotted lines indicate the baseline potential, the peak of the action potential, and the voltage level at 50% repolarization of the action potential. The action potential duration was measured as the time from the start of the upstroke ({downarrow}) to 50% repolarization of the peak ({uparrow}).

 

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  		Table 2. APD50 of isolated perfused hearts from control and SNx ratsa
 


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Renal function in the partially nephrectomized rats was significantly impaired 8 wk after surgery (e.g., threefold reduction in creatinine clearance) (Table 1). The impairment of renal function was associated with myocardial hypertrophy, particularly of the left ventricle and septum, and histologic evidence of myocardial fibrosis in the subendocardial region of the left ventricular wall (Figure 2), as has been reported in this model (15,16). Because the systolic BP was not increased in the SNx rats, this cardiomyopathy was not the result of increased afterload. LVH in the absence of increased BP in SNx rats has been reported (15). In the present study, the cell capacitance was not significantly different between myocytes from SNx and control rats, suggesting that there was no increase in the membrane surface area of myocytes in the SNx model. However, other groups have reported small, although statistically nonsignificant, increases in cell size in SNx myocytes (17).

The 4-AP-sensitive K+ current Ito is believed to play an important role in modulating cardiac excitability and conduction and in early phase repolarization of the action potential (21,22,23,24,25,26,34). The properties of Ito were examined in myocytes from SNx and control hearts. In our experiments, Ito was activated by potentials from -30 mV to positive (Figure 3), was completely blocked by 4-AP, and showed voltage-dependent inactivation (Figure 6), consistent with other reports of Ito in rat ventricular myocytes (21,22,33). In contrast, Iso was relatively insensitive to 4-AP and did not show voltage-dependent inactivation, confirming that Ito was distinct from Iso. Unlike Ito, Iso showed little time-dependent inactivation during voltage pulses, as can be seen after inactivation of Ito by prepulses to positive potentials (Figure 5). Taken together, these findings justify the measurement of Ito as the difference between Ipk and Iso. Differences in experimental conditions (e.g., temperature, presence of divalent cation L-type Ca2+ channel blockade) make direct quantitative comparison of our results with other reports difficult (35,36). However, the voltage of half-maximal inactivation of Ito of -50 mV in the present study was within the range reported by other groups (-60 to -29 mV) (21,22,27,28,36). Ito inactivated at positive potentials with a time constant ({tau}fast) of approximately 10 ms. In 33% of the cells, an additional slower exponential component could be observed with a time constant of around 70 ms. This slow component of inactivation was relatively small (approximately 12% of total current) and poorly resolved during the 200-ms pulse. In the present study, the time course of recovery of Ito from inactivation was described by a single exponential with a time constant of approximately 13 ms in control myocytes (Figure 7). The currents were not fully recovered at the longest interpulse intervals used (75 ms). There have been reports very recently that the recovery from inactivation of Ito in rat ventricular myocytes can be described as the sum of two exponential components ({tau}fast approximately 25 ms, {tau}slow >180 ms), and these data are consistent with the present study (37,38). Longer interpulse intervals may have allowed us to resolve a slower component of recovery from inactivation in the present study.

The slowly inactivating component to Ito may correspond with the slowly inactivating currents recently identified in rat (IKx) and mouse (Ito,s) ventricular myocytes (39,40). It has been suggested that the rapidly inactivating and rapidly recovering component to Ito in rat ventricular myocytes corresponds to Kv4.2/Kv4.3 voltage-gated K+ channel subunits, whereas the slowly inactivating, slowly recovering component corresponds to Kv1.4 (38). Other groups have suggested that Kv1.2 may contribute to the slowly inactivating current (39).

Heterogeneity of Ito in ventricular myocytes is well recognized (22,23,26,27,38,39,40,41,42), and differences in Ito have been reported both between different regions of the ventricle and between cells within the same region (23,26,27,38,39,40,41,42). A gradient of Ito density has been reported not only from the base of the left ventricle to the apex but also from left ventricle endocardium to epicardium and between the septum and the right ventricle (23,27,38,39,40,41,42). Heterogeneous expression of Kv4.2, Kv4.3, and Kv1.4 channel subunits may underlie the regional differences in Ito (38,42,43,44). The heterogeneous distribution of Ito within the ventricles is believed to contribute to regional differences in the action potential duration and may play an important role in the physiologic regulation of excitation and conduction and the sequence of ventricular repolarization (23,26,41,45,46,47). Thus, Ito density tends to be highest, and action potential duration shortest, in the epicardial layer, in the apex, and in the right ventricle. The regional differences in APD50 recorded from the epicardial surface of isolated perfused hearts in the present study were consistent with the expected heterogeneity in Ito (Table 2). Because myocytes were not isolated from distinct regions of the ventricle in the present study, regional differences in Ito density are likely to have contributed to the observed heterogeneity in Ito (Figure 4).

SNx was associated with striking abnormalities in the transient outward current Ito, which was more than doubled in size in SNx myocytes compared with controls. Neither Iso nor the inward rectifier K+ current IK1 was altered. Ito was increased at all voltages from -30 to +60 mV in SNx myocytes compared with controls, and the heterogeneity between cells was increased (Figures 3 and 4). In addition, the time course of the onset of inactivation of Ito and its recovery were prolonged in SNx myocytes compared with controls, associated with an increase in {tau}fast (Figure 6) and {tau}rcc (Figure 7). Taken together, an increase in the amplitude of Ito and slowing of the onset of inactivation might be expected to result in shortening of the action potential duration in renal failure. The APD50 at the base of the LV of isolated perfused hearts was shortened by about 20% after SNx (Table 2). The APD50 values in hearts from both SNx and control rats were prolonged by 2 mM 4-AP, illustrating the importance of a 4-AP-sensitive current to normal repolarization. Moreover, 2 mM 4-AP abolished the differences between hearts from control and SNx hearts in the APD50 at the base of the LV, strongly suggesting that the changes in 4-AP-sensitive Ito contributed to the APD50 shortening in renal failure. Thus, were the increased density and heterogeneity in Ito in SNx observed in the present study to occur in humans, it would be likely to result in abnormalities in cardiac excitation and conduction and increased susceptibility to ventricular dysrhythmias, particularly re-entrant tachycardias (26).

The changes in Ito of the present study, produced without increases in BP, are in marked contrast to those reported in abdominal aortic banding models of LVH associated with increased afterload, in which both Ito and the regional heterogeneity in Ito are reduced (27,28). Reductions in Ito density have also been reported in spontaneously hypertensive and deoxycorticosterone acetate-induced models of hypertension (29,30). Thus, cardiac myocytes show abnormalities in Ito in SNx rats distinct from those reported in hypertension, emphasizing the fact that a renal lesion causes abnormalities in the electrophysiologic properties of cardiac myocytes independent of changes in BP. A model of renovascular hypertension, Goldblatt "2-kidney, 1-clip," showed an increase in Ito and a lengthening of the {tau} of inactivation of K+ currents (31) similar to those found in the present study. Because the changes in Ito in the Goldblatt model are quite different from those reported in other models of hypertension, it seems likely that the increase in Ito was independent of changes in BP and may have been linked to the renal ischemia characteristic of this model.

The properties of cardiac Ito are known to be modulated under other pathologic conditions, including hypothyroidism, hyperthyroidism, and diabetes (48,49). For example, ventricular myocytes from thyroidectomized rats and streptozotocin-induced diabetic rats showed reduced current density and reduced regional heterogeneity in Ito (48). On the other hand, hyperthyroidism was associated with an increase in Ito density in rabbit ventricular myocytes (49) and an increase in the rate of recovery from inactivation of Ito in rat ventricular myocytes (48), suggesting a role for thyroid hormone in the regulation and expression of cardiac Ito channels. The expression and function of Ito channels are regulated by paracrine and circulating hormones. For example, the expression of Shal-type K+ channel {alpha}-subunits (Kv4.2 and Kv4.3), believed to underlie cardiac Ito, in neonatal rat ventricular myocytes is regulated by thyroid hormone (50). Paracrine factors produced by nonmyocyte cardiac cells downregulate Ito and the expression of K+ channel {alpha}-subunits in cultured rat cardiac myocytes (51).

In principle, the increased current density of Ito observed in the present study may result from an increase in the number of channels or an alteration in the properties of the channel proteins (i.e., gating, single channel conductance). Channel properties might be altered in renal failure through changes in posttranslational modification or regulation. For example, it has been suggested that translocation of the {epsilon}-isoform of protein kinase C contributes to the changes in Ito in myocytes from hypothyroid and diabetic rats (52). It is unclear what changes in K+ channel expression or function underlie the changes in Ito in renal failure. However, because the rapidly inactivating and rapidly recovering component to Ito predominated in our experiments, it seems likely that changes in the properties of Kv4.2 or Kv4.3 channels contributed to the changes in Ito observed. Direct immediate effects of uremic toxins or changes in plasma electrolyte levels on Ito cannot account for the changes after SNx, because the experiments were performed on isolated cells or isolated hearts perfused with a uremic toxin-free experimental solution. The mechanism by which ventricular Ito was altered in the present study may involve indirect effects of uremic toxins, indirect effects of changes in plasma electrolyte levels, or a hormonal signal from the remnant kidney. Alternatively, anemia or secondary hyperparathyroidism may play a role. Iron deficiency models of anemia in the rat have been shown to result in ventricular hypertrophy and shortening of the action potential duration, although the properties of K+ currents have not been examined in this model (53,54). Studies of cardiac myocyte ion currents in rat models of renal failure with correction of anemia and/or parathyroidectomy would help shed further light on this.


   Acknowledgments
 
This work was funded by the British Heart Foundation (Ph.D. studentship to Dr. Donohoe).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication August 31, 1999. Accepted for publication February 28, 2000.




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G.-X. Yan, S. J. Rials, Y. Wu, T. Liu, X. Xu, R. A. Marinchak, and P. R. Kowey
Ventricular hypertrophy amplifies transmural repolarization dispersion and induces early afterdepolarization
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