Department of Renal Medicine, GKT School of Medicine, King's College London, 1 Anthony Raine Research Laboratories, St. Bartholomew's Hospital, Queen Mary Westfield College, 2 Cardiac Surgical Research and 3 Cardiovascular Research, The Rayne Institute, Centre for Cardiovascular Biology and Medicine, St. Thomas' Hospital, King's College London, London, UK
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Abstract |
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Methods. Wistar rats underwent two-stage SNx; control rats (C) underwent bilateral renal decapsulation. Animals were sacrificed after 8 weeks, and ventricular myocytes were isolated. SNx rats showed a 2-fold increase in plasma urea and creatinine compared with C rats. Whole-cell patch clamp techniques were used to examine L-type Ca2+ channel currents in isolated cardiac myocytes at 37°C. In separate experiments, the epicardial monophasic action potentials of isolated perfused whole hearts from C and SNx rats were recorded.
Results. The amplitude and currentvoltage relationships of the L-type Ca2+ current were not significantly different in myocytes from C (n=11) and SNx (n=8) rats. However, the rate of inactivation of the Ca2+ current was increased by ~1525% (P<0.05) in myocytes from SNx rats. The action potential duration (APD33) at the apex of the left ventricle was ~20% shorter (P<0.01) in hearts from SNx rats as compared with controls.
Conclusions. Renal failure is associated with rapid inactivation of cardiac ventricular myocyte L-type Ca2+ currents, which may reduce Ca2+ influx and contribute to shortening of the action potential duration.
Keywords: cardiac action potential duration; isolated cardiac myocytes; L-type Ca2+ currents; partial nephrectomy model; uraemic cardiomyopathy; whole-cell patch-clamp
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Introduction |
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It is clear from animal work that renal failure is associated with cardiac contractile dysfunction, even at the level of the single cell [7,8]. Previous work using subtotal nephrectomy (SNx) models of chronic uraemia in the rat has demonstrated a 25% reduction in cardiac output of isolated perfused hearts from SNx rats compared with hearts from sham-operated controls [7]. More recently, isolated ventricular myocytes from the same animal model have been studied. A reduction in both the amplitude and velocity of contraction of isolated cardiac myocytes from SNx rats has been demonstrated [8]. Both the reduction in cardiac output and the reduction in contractile amplitude of isolated cardiac myocytes following SNx were associated with a reduced positive inotropic response to external Ca2+, suggesting a possible change in Ca2+ handling by cardiac myocytes following SNx [7,8]. We previously have examined systolic and diastolic Ca2+ levels in electrically paced isolated cardiac myocytes in SNx and control rats. While diastolic Ca2+ levels appeared to be elevated in myocytes from SNx rats, there was no change in the maximum systolic Ca2+ level [911]. Thus, SNx appears to be associated with abnormalities of Ca2+ handling and excitation contraction coupling in the rat subtotal nephrectomy model.
Here we present data using whole-cell patch-clamp techniques to examine the L-type Ca2+ currents of isolated cardiac ventricular myocytes in the subtotal nephrectomy model of chronic renal failure in the rat. L-type Ca2+ channels are responsible for the vast majority of Ca2+ influx into cardiac myocytes following depolarization, and may be one of the components of excitationcontraction coupling affected by SNx. We also examined the effect of SNx on epicardial monophasic action potential duration in isolated perfused hearts.
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Methods |
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Myocyte isolation
Ventricular myocytes were isolated by retrograde perfusion of the heart via the aorta with an enzyme-containing solution (collagenase class 2, Worthington, USA and hyaluronidase I-C, Sigma, UK), as previously described [8]. Cells were stored in 100 µM Ca2+-containing solution at room temperature, and used in patch-clamp experiments within 8 h of isolation.
Electrophysiological experiments
Isolated cells were perfused with Tyrode solution (in mM: NaCl 142.0, KCl 5.4, NaH2PO4 0.4, MgCl2 1.1, HEPES 10.0, glucose 5.5, taurine 10.0, CaCl2 1.0, pH 7.4 with NaOH) at 37°C and a flow rate of 23 ml/min. Myocytes suitable for patch-clamp experiments were Ca2+-tolerant, single, rod-shaped cells that ranged from 20 to 50% of isolated cells. Pipettes had a tip resistance of 24 M when filled with the Cs+-rich pipette solution (in mM: L-aspartic acid 85, CsOH 85, TEA-Cl 20, HEPES 10, EGTA 1, MgCl2 2, MgATP 4, Na2GTP 0.1, Na2 creatine phosphate 5, pH adjusted to 7.4 with TEA-OH). In some experiments, the EGTA concentration of the pipette solution was 10 mM, as indicated in the text.
Whole-cell patch-clamp recordings were made using EPC-9 (HEKA GmbH, Germany) and Axopatch 200B (Axon Instruments Inc., USA) amplifiers. Data were acquired at 2 kHz to the hard disc of an Apple computer using Pulse/PulseFit software (HEKA GmbH, Germany). Series resistance and capacitance compensation were applied electronically. Voltage pulses (250 ms) between -60 and +60 mV were applied in 10 mV increments from a holding potential of -60 mV every 4 s (Figure 1A). Pre-pulses (30 ms) to -40 mV were used to inactivate voltage-dependent Na+ channels. Paired pulse protocols were applied to quantify the steady-state voltage dependence of Ca2+ channel inactivation. A series of 300 ms pre-pulses were applied every 4 s from -80 to +40 mV in 5 mV increments, followed by a 200 ms test pulse to -10 mV.
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All data are expressed as mean±SE. Statistical significance was assessed by either two-tailed ANOVA or Student's t-test, and P<0.05 was considered significant. Curve fitting was performed using IgorPro Vers 2.1.
Epicardial monophasic action potentials
Control and SNx rats were prepared surgically as before. A total of five control and seven SNx rat hearts were used to record monophasic action potentials (MAPs), again at 8 weeks following the second surgical procedure.
Rats were anaesthetized with SalanaxTM (Pentobarbitone, Rhone Merieux) 0.1 ml/100 g intraperitoneally, and heparinized with 0.15 ml of 1 : 5000 heparin solution (Leo Laboratories, UK). Depth of anaesthesia was assessed by hind leg withdrawal reflex. Hearts were excised into ice-cold KrebsHenseleit buffer (in mM: NaCl 119, KCl 4.7, MgSO4 1.2, H2PO4 1.2, NaHCO3 25, glucose 11.5, CaCl2 1.2 mM, pH 7.4 with 1 mM NaOH, bubbled with 95% O25% CO2 mix) and mounted on a Langendorff perfusion apparatus. Perfusion was commenced with KrebsHenseleit buffer at 37°C in a retrograde manner with a perfusion pressure of 100 cmH2O, resulting in flow rates ranging from 10 to 15 ml/min. Hearts were unpaced and were allowed to beat spontaneously.
After a 20 min stabilization period, monophasic action potentials were recorded using an epicardial suction electrode, positioned in turn at the apex and base of the left ventricle, and at the right ventricle.
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Results |
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Voltages from -30 mV to positive elicited rapidly activating and inactivating, time-dependent inward currents (Figure 1A) which were blocked by bath application of 0.1 mM CdCl2 (Figure 1B
), consistent with the properties of cardiac L-type Ca2+ currents.
There was no significant change in the amplitude and currentvoltage relationship of Ca2+ current following SNx (Figure 1C). The currentvoltage relationship was U-shaped, and generally maximally inward at -10 mV for both control (-16.8±2.0 pA/pF) and SNx (-18.3±3.1 pA/pF) myocytes. There was also no difference in mean time-to-peak of Ca2+ current between SNx and C myocytes (4.6±0.5 and 4.9±0.5 ms at 0 mV respectively, Figure 2A
).
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Neither f nor
s showed significant voltage dependence between -10 and +10 mV (Figure 3A
). SNx was associated with shortening of both
f (C 17±0.8 ms; SNx 15±1.6 ms at 0 mV) and
s (C 57±4.0 ms, SNx 46±2.0 ms at 0 mV) for all voltages from -10 to +10 mV (Figure 3A
). The shortening of the slow time constant (
s) was significant (P<0.05). SNx had no effect on the values of A1 or A2 (Figure 3B
), and it was clear that the slow component of inactivation predominated over the fast component, as A2 was greater in magnitude than A1 for all voltages between -10 and +10 mV.
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The duration of the action potential in isolated perfused hearts from C (n=5) and SNx (n=7) animals was examined by monophasic action potential recordings from the epicardial surface of the ventricle. Figure 5 shows example recordings of monophasic action potentials 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 33% repolarization of the peak of the action potential (APD33), are shown in Table 1
. APD33 was shorter in the LV apex and RV as compared with the LV base. SNx shortened the APD33 by ~20% in the LV apex (P<0.05). There was no significant difference between SNx and control animals in the heart rate of isolated perfused hearts.
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Discussion |
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Cardiac L-type Ca2+ channels inactivate by Ca2+-and voltage-dependent mechanisms [18]. Ca2+-dependent inactivation ( ~50 ms) is very much faster than voltage-dependent inactivation (
~500 ms), and predominates under physiological conditions when Ca2+ is the principle charge carrier [18]. Ca2+-dependent inactivation occurs as a result of Ca2+ binding to the
1c subunit of the channel in the micromolar range [19]. Calmodulin may contribute to the Ca2+-dependent inactivation of the L-type Ca2+ current [20]. Thus, the inactivation rate is dependent on the Ca2+ concentration in the immediate vicinity of the channel at the cytosolic surface of the membrane and is highly sensitive to Ca2+ influx through the channel and Ca2+ release from the sarcoplasmic reticulum [17]. Ca2+-dependent mechanisms most probably contributed to the L-type current inactivation reported here since Ca2+ ions were the principal charge carrier. However, increasing the intracellular Ca2+ buffering capacity by changing the pipette [EGTA] from 1 to 10 mM did not slow inactivation significantly and did not reveal the much slower voltage-dependent mechanism. Thus, even with 10 mM EGTA in the pipette, it was not possible to chelate cytosolic Ca2+ in the vicinity of the channel sufficiently to prevent Ca2+-dependent inactivation. This is consistent with the findings of other groups who also show Ca2+-dependent inactivation when 10 mM EGTA was used as the Ca2+ buffer in the pipette [21].
The mechanism of the effect of SNx on L-type Ca2+ current inactivation is unclear. Previous work has shown an increase in basal cytosolic Ca2+ concentration ([Ca2+]c) associated with SNx in isolated ventricular myocytes [9,10]. Since Ca2+-dependent inactivation is dependent on micromolar range changes in [Ca2+] in the locality of the channel, it is unlikely that these changes (C ~60 nM; SNx ~100 nM) in basal [Ca2+]c in the nanomolar range could have resulted in the speeding up of inactivation seen in our experiments. Since neither the peak Ca2+ current nor systolic Ca2+ release [10] are increased after SNx, it is unlikely that an increase in the [Ca2+]c at the cytosolic surface of the Ca2+ channel upon excitation contributes to the changes in inactivation in renal failure. Rather, it seems likely that a change in the Ca2+ sensitivity of the inactivation mechanism results in an increased rate of Ca2+ current inactivation after SNx.
The shortening of inactivation of the L-type Ca2+ current may be due to altered channel protein conformation or function as a result of changes in protein expression or post-translational modification. Recent evidence suggests the existence of alternative splice variants of the cardiac L-type Ca2+ channel 1c subunit and changes in the expression of splice variants in cardiomyopathy [21]. Changes in the expression of alternative splice variants of the third membrane-spanning domain of motif IV (or IVS3) of the
1c subunit have been shown in a model of myocardial infarction-induced hypertrophy in the rat [22]. Other splice variant isoforms of the
1c protein differing in domains known to be important in voltage- (IS6) and Ca2+-dependent (C-terminal cytosolic tail) inactivation mechanisms have been reported [23,24]. Thus, one explanation for the increased rate of inactivation might be the expression of splice variants in SNx experiments, particularly variants differing in the C-terminus of the
1c subunit. Alternatively, changes in binding of calmodulin may contribute to the changes following SNx [20].
Rapid inactivation of the Ca2+ current in uraemia can be expected to have two consequences for cardiac function: firstly, a reduction in the total amount of Ca2+ influx during depolarization. The observed shortening of the mean time constants of inactivation reduced the estimated total Ca2+ influx by 22%, from 4.62x10-7 mol/cm2 to 3.59x10-7 mol/cm2. A reduction in the Ca2+ influx after SNx might be expected to result in a reduced Ca2+ content of the sarcoplasmic reticulum and, thereby, less Ca2+ release on stimulation. However, we have shown previously that the Ca2+ transient was not affected and the resting cytosolic Ca2+ concentration was increased after SNx [911], an effect that cannot be accounted for by reduced Ca2+ influx through L-type Ca2+ channels. Taken together, these facts suggest that changes in other Ca2+ handling pathways (e.g. Na+/Ca2+ exchange, sarcoplasmic reticular Ca2+ sequestration, Ca2+ buffering) may be contributing to the abnormalities in Ca2+ handling in uraemia.
The epicardial action potential duration at 33% repolarization from the apex of the left ventricles was found to be significantly shortened by ~20% in hearts from SNx rats compared with C rats (Table 1). Rapid inactivation of the Ca2+ current may contribute to this action potential shortening after SNx. The action potential duration determines the refractory period and is important in maintaining normal rhythmic cardiac function. We have found recently that changes in the properties of a 4-aminopyridine-sensitive repolarizing K+ current may also contribute to changes in the action potential duration in renal failure [25]. Accordingly, the changes in ion currents, Ca2+ handling and excitationcontraction coupling of isolated cardiac myocytes in renal failure are the subject of further investigation.
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Acknowledgments |
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Notes |
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References |
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