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1 Institut für Physiologie, Universitätskrankenhaus Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 2 University Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK 3 Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraße 6, 91054 Erlangen, Germany
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Abstract |
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(Received 20 July 2004;
accepted after revision 27 September 2004; first published online 4 October 2004)
Corresponding author T. Volk: Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Waldstraße 6, 91054 Erlangen, Germany. Email: tilmann.volk{at}physiologie2.med.uni-erlangen.de
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Introduction |
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A decrease in the density of Ito has been observed in many experimental models of cardiac hypertrophy and failure (reviewed by Kääb et al. 1998) as well as in end-stage cardiac failure in humans (Beuckelmann et al. 1993), and contributes to the altered shape of the AP in hypertrophied or failing ventricles. We have recently shown that cardiac hypertrophy induced by ascending aortic stenosis (AS) predominantly increases the APD in epicardial myocytes of the rat left ventricular free wall and identified a reduction in Ito in epicardial myocytes as the major underlying alteration in ionic currents (Volk et al. 2001). Although density and kinetics of the L-type Ca2+ current (ICaL) are unaffected by AS (Volk & Ehmke, 2002), the altered AP profile is likely to influence AP-induced Ca2+ currents. Indeed, representative APs obtained from normal and diseased rats (myocardial infarction) have previously been used to assess the influence of an altered AP-shape on AP-induced Ca2+ currents and revealed a significant increase in the amplitude of the Ca2+ transient and unloaded cell shortening (Kaprielian et al. 1999; Sah et al. 2001). However, in these studies the profound regional differences in Ito magnitude (Clark et al. 1993; Bénitah et al. 1993; Volk et al. 1999) in the left ventricle were not taken into account, thus potentially ignoring the possibility that effects of cardiac diseases may be limited to specific regions of the heart, thereby affecting the regional organization of the ventricle. Moreover, in addition to regional differences, there may even be a considerable cell-to-cell variability in the density of ionic currents, especially of Ito and ICaL (Gómez et al. 1997), which strongly determine the individual shape of each cell's AP.
In the present study, we therefore used the AP voltage-clamp technique to directly assess the effect of cardiac hypertrophy on AP-induced Ca2+ influx in individual endo- and epicardial myocytes of the rat left ventricular free wall. On the basis of these experimental data and a recently developed electrical model of rat epicardial myocytes, we estimated to what extent a decrease in Ito alone can account for the observed effects of hypertrophy on AP shape and duration and AP-induced Ca2+ influx. In addition, a possible contribution of alterations in L-type channel gating to the altered AP-induced Ca2+ current was investigated.
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Methods |
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Ascending aortic stenosis (AS) was induced as previously described (Wiesner et al. 1997). Briefly, female Sprague–Dawley rats weighing 180–190 g were anaesthetized by I.P. injection of a mixture of ketamine HCl and xylazine HCl (100 and 4 mg kg–1 body weight, respectively), intubated and mechanically ventilated. The ascending aorta was partly occluded by a Hemoclip (Pilling Weck Inc., Research Triangle Park, NC, USA) set to an outer diameter of 0.85 mm. In sham-operated animals, the aortic root was occluded for 5–10 s with forceps instead of clipping. Mortality resulting from the surgical intervention was <10%. All animal experiments were conducted in accordance with institutional guidelines and approved by local authorities.
Haemodynamic measurements
Seven days after surgery the rats were anaesthetized by I.P. injection of Trapanal (thiopentone sodium, Byk Gulden, Konstanz, Germany), 100 mg (kg body weight)–1. A femoral artery catheter was inserted to measure peripheral blood pressure. Left ventricular pressure was measured by cannulating the left ventricle through the cardiac apex. Blood pressure was sampled at 200 Hz using a DAS 1602 interface (Keithley Instruments Inc. Taunton, MA, USA) connected to a Pentium-based PC and controlled by LabTech Notebook pro software (Labtech, Wilmington, MA, USA).
Isolation of myocytes
After completion of the haemodynamic measurements the heart was quickly excised and placed into cold (4°C) cardioplegic solution, where it stopped beating immediately. The stenosing clip was removed, the heart was mounted on a Langendorff apparatus and left ventricular myocytes of subendocardial and subepicardial origin were isolated according to the method previously described (Isenberg & Klöckner, 1982; Volk & Ehmke, 2002). Briefly, the aorta was cannulated and retrogradedly perfused with nominally Ca2+-free modified Tyrode solution at 37°C for 5 min. Perfusion pressure was 75 mmHg, and all solutions were equilibrated with 100% oxygen. The perfusion was continued for 15 min with 20 ml of the same solution containing collagenase (type CLS II, 200 U ml–1, Biochrom KG, Berlin, Germany) and protease (type XIV, 0.7 U ml–1, Sigma Chemical Co., St Louis, MO, USA), and the solution was recirculated. Finally, the heart was perfused with modified Tyrode solution containing 100 µM Ca2+ for another 5 min. After the perfusion, the left ventricular free wall was separated from the rest of the heart, and endo- and epicardial tissue was carefully isolated from the left ventricular free wall with fine forceps and placed into separate cups. To further disaggregate the tissue pieces, they were gently shaken at 37°C for several minutes, filtered through a cotton mesh and allowed to settle for half an hour. Cells were stored at room temperature in modified Tyrode solution containing 100 µM Ca2+. Only single rod-shaped cells with clear cross-striations and no spontaneous contractions were used for experiments.
Solutions and chemicals
Cardioplegic solution contained (mM): NaCl, 15; KCl, 9; MgCl, 4; NaH2PO4, 0.33; CaCl2, 0.015; glucose, 10; and mannitol, 238; titrated to pH 7.40 with NaOH. Gigaohm seals were obtained in modified Tyrode solution (control solution; mM): NaCl, 138; KCl, 4; MgCl2, 1, NaH2PO4, 0.33; CaCl2, 2; glucose, 10; and Hepes, 10; titrated to pH 7.30 with NaOH. For AP voltage-clamp experiments we used the same pipette solution as we have used previously (Volk et al. 1999) to allow for comparison of the results. It contained (mM): glutamic acid, 120 (titrated with KOH and thus resulting in K-glutamate, 120); KCl, 10; MgCl2, 2; EGTA, 10; Na2-ATP, 2; and Hepes, 10; titrated to pH 7.20 with KOH. The bath solution in these experiments was control solution.
Patch clamp technique
The ruptured-patch whole-cell configuration was used as previously described (Hamill et al. 1981). Myocardial cells were transferred in an elongated chamber (2.5 x 20 mm), mounted on the stage of an inverted microscope (Axiovert 25, Zeiss, Jena, Germany) and initially superfused with control solution. All experiments were performed at room temperature (22–26°C). Patch pipettes were pulled from borosilicate glass (GC150-15, Clark Electromedical Instruments, Reading, UK) using a P-87 Puller (Sutter Instruments, Novato, CA, USA). Pipette resistance (RPip) averaged 3.1 ± 0.1 M
(n = 133) with K-glutamate in the pipette and control solution in the bath.
Currents were recorded using an EPC-9 amplifier (HEKA Electronik, Lambrecht, Germany), controlled by a Power-Macintosh (Apple Computer, Cupertino, CA, USA) and PULSE-Software (HEKA Elektronik). Membrane voltage (Vm) and APs were recorded in the zero current-clamp mode and ionic currents in the voltage-clamp mode. For AP voltage-clamp recordings, APs were recorded at the beginning of the experiments and used as a voltage template in the voltage-clamp mode of the amplifier (Doerr et al. 1989). Membrane capacitance (Cm) and series resistance (Rs) were calculated using the automated capacitance compensation procedure of the EPC-9 amplifier. Series resistance averaged 5.1 ± 0.1 M (n = 133) and was compensated by 85%, leading to an average effective Rs of 
0.8 M. Accordingly, at the largest recorded currents of interest, which were about 3 nA, the voltage error was less than 3 mV. In myocytes isolated from sham-operated animals, Cm averaged 116 ± 3 pF (n = 65) and was similar in both endo- and epicardial myocytes. In myocytes isolated from AS rats, Cm increased to a similar extent in endo- and epicardial myocytes and averaged 160 ±5 pF (n = 68, P < 0.0001 vs. sham), demonstrating hypertrophy at the cellular level. The reference electrode of the amplifier headstage was bathed in pipette solution in a separate chamber and was connected to the bath solution via an agar-agar bridge filled with pipette solution. Pipette potential (VPip) and Vm were corrected for liquid junction potentials at the bridge–bath junction (13.3 mV for K-glutamate pipette solution and control solution in the bath). Whole-cell currents were low-pass filtered at 1 kHz and sampled at 5 kHz. Action potentials were sampled at 1 kHz. Whole-cell data were analysed using PULSE-FIT software (HEKA Elektronik) and IGOR Pro (WaveMetrics, Lake Oswego, OR, USA). Data are given as means ± S.E.M. Statistical significance was calculated by a one-way ANOVA followed by a Bonferroni post hoc test using the software PRISM (Graph-Pad Inc., San Diego, CA, USA). In cases where only two groups were compared (e.g. haemodynamic measurements), the appropriate version of Student's t test was used. Differences with P < 0.05 were considered statistically significant.
Identification and separation of currents
Using the AP voltage-clamp method, AP-induced Ca2+ currents and Ca2+ influx were estimated by subtraction of recordings made in the presence of 300 µM Cd2+ in the bath solution (control solution) from recordings without Cd2+. This difference represents the Cd2+-sensitive current. Cd2+ at a concentration of 300 µM is a relatively non-specific blocker of Ca2+ channels (Fox et al. 1987) and may also affect other ionic currents, e.g. the steady-state activation and inactivation properties of Ito (Agus et al. 1991) and the Na+–Ca2+ exchange current. However, more specific inhibitors of ICaL, such as dihydropyridines or D600, are also potent inhibitors of Ito (Gotoh et al. 1991; Lefevre et al. 1991) and would thus interfere with the estimation of QCa in the presence of K+ currents. Since the pipette solution contained 10 mM of the Ca2+ chelator EGTA, Ca2+-dependent currents, such as the Ca2+-activated Cl– current (Zygmunt & Gibbons, 1992), would be prevented from activation. Furthermore, in the absence of intracellular Ca2+, the Na+–Ca2+ exchanger does not contribute to the whole-cell current (Allen & Baker, 1985). Despite these limitations, the AP-induced Cd2+-sensitive current does yield a reasonable estimate for the AP-induced Ca2+ current and has been used previously (Puglisi et al. 1999; Volk et al. 1999).
Model calculations
The model of a rat ventricular epicardial myocyte published by Pandit et al. (2001) was coded into and run using CellEditor (Physiome Sciences Inc., Princeton, NJ, USA). This model was chosen mainly for two reasons: first, the model is the rat model which was developed most recently and therefore includes recent advances in rat cellular electrophysiology; and second, the model published by Pandit et al. (2001) does take endocardial–epicardial differences into account, thus making it specifically advantageous for the purpose of present study. The stimulus current used was 0.6 nA for a period of 5 ms at a frequency of 1 Hz. Similar results were obtained also when a lower stimulus rate of 0.3 Hz was used (data not shown). For AP clamp simulations the calculated membrane voltage was used as a voltage template to clamp the model. The model calculation included the presence of 10 mM EGTA in the pipette solution. The decrease in Ito magnitude was simulated by setting the conductance parameter (Gt) to 70% of the control value and the decrease in the magnitude of the inwardly rectifying K+ current (IK1) was simulated accordingly.
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Results |
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In sham-operated animals, left ventricular peak pressure and peripheral systolic blood pressure were nearly identical (123 ± 5 vs. 121 ± 5 mmHg, n = 9). Ascending aortic stenosis led to a significant pressure difference between the left ventricle (170 ± 10 mmHg) and the femoral artery (86 ± 6 mmHg, n = 8, P < 0.0001) of 84 ± 7 mmHg. In previous studies, in which rats of the same gender and similar breed and age were used, we have shown that a similar increase in afterload induces a 30% increase in the relative left ventricular weight (Wiesner et al. 1997; Moser et al. 2002).
Action potentials and AP-induced Ca2+ influx
To investigate the effect of hypertrophy on AP-induced Ca2+ current, we used the AP voltage-clamp technique (Doerr et al. 1989). Action potentials were recorded at the beginning of each experiment in the current-clamp mode of the amplifier at a stimulus rate of 0.3 Hz. Then, after switching to the voltage-clamp mode, Vm of individual myocytes was clamped to their own previously recorded AP, and the AP-induced current was recorded in the absence and presence of 300 µM Cd2+ in the bath solution. Subtraction analysis yielded the Cd2+-sensitive current, which gives an estimate for the AP-induced Ca2+ current. Figure 1 shows average recordings of all APs and AP-induced Ca2+ currents recorded from endo- and epicardial myocytes of sham-operated (Fig. 1A) and AS animals (Fig. 1B). Average APs and Ca2+ current traces were calculated after shifting all APs with reference to their individual peaks, the maximum shift necessary being 10 ms. The corresponding AP-induced Ca2+ currents were shifted to the same extent. Capacitive current components and incompletely subtracted fast Na+ currents that occurred before the AP-induced Ca2+ current were removed from the average current traces for clarity. In sham-operated rats, APs recorded from epicardial myocytes were substantially shorter and displayed much more pronounced phase 1 repolarization than endocardial myocytes. Consequently, APD0mV (i.e. APD at repolarization to 0 mV) and APD90 (i.e. APD at 90% repolarization) were significantly longer in endocardial myocytes (see Table 1). These results correspond very well to those that we have recorded previously from myocytes of untreated rats (Volk et al. 1999), indicating that sham operation has little or no influence on cardiac electrophysiology. Action potentials recorded from endocardial myocytes of AS animals were very similar to those of sham-operated animals. In contrast, APs recorded from epicardial myocytes of AS animals were significantly prolonged, especially at membrane potentials negative to a Vm of 0 mV. Accordingly, the APD90 significantly increased in epicardial myocytes of AS animals. There was a slight increase of APD0mV, but this effect did not reach statistical significance (see Table 1).
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Time course of AP-induced Ca2+ influx
Figure 2 illustrates the time course of the AP-induced Ca2+ influx by plotting the cumulative average QCa, as observed in each individual myocyte, vs. time. In endocardial myocytes, AS had virtually no effect on the cumulative QCa. In contrast, in myocytes of epicardial origin, AS displayed a pronounced increase in the AP-induced Ca2+ influx after 40 ms from the beginning of the AP and eventually reached twice the magnitude of sham-operated rats.
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–5 mV in epicardial myocytes and Vm 20 mV in endocardial myocytes. At a Vm of 20 mV the driving force for Ca2+ influx is substantially lower than at a Vm of –5 mV. Nevertheless, the peaks of the AP-induced Ca2+ currents were of similar magnitude in endo- and epicardial myocytes because of the differences in the conductance of the L-type Ca2+ current observed at the peaks of the AP-induced Ca2+ currents (GPeak). In epicardial myocytes of both sham-operated and AS animals, GPeak was significantly lower than in endocardial myocytes. Since there was no significant effect of AS on GPeak, the data of sham-operated and AS animals were pooled (200 ± 14 pS pF–1, n = 80 in epicardial myocytes vs. 252 ± 15 pS pF–1, n = 53, P < 0.05 in endocardial myocytes). The difference in GPeak indicates that the differences in AP waveforms between endo- and epicardial myocytes lead to altered channel gating behaviour, resulting in a lower conductance at the peak of the AP-induced Ca2+ current in epicardial myocytes. Ascending aortic stenosis had only a small influence on the profile and the conductance of the AP-induced Ca2+ current, but markedly altered its time course. This is illustrated by the arrows in Fig. 3, which depict the time at which certain points of the current–voltage relation were reached during the AP. In epicardial myocytes of sham-operated rats, for example, Vm returned to values below –40 mV (a potential below which the Ca2+ influx decreased to very low levels) after an average of 44 ± 11 ms, whereas in epicardial cells of AS animals it took 124 ± 18 ms for Vm to decrease to below –40 mV (P < 0.01). Consequently, in epicardial myocytes of AS animals, a significant Ca2+ influx was maintained over a much longer period during the AP, resulting in the increase in QCa observed in this group. In contrast, in endocardial myocytes there was no significant difference in the time until Vm returned to values below –40 mV (222 ± 22 ms in sham-operated vs. 225 ± 22 ms in AS animals).
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The experimental data presented in this study demonstrate a significant increase in QCa in epicardial myocytes in response to AS, while in endocardial myocytes, APs and QCa were largely unaffected. The increase in QCa in epicardial myocytes appears to result from an increased APD, especially in the membrane potential range which facilitates a significant Ca2+ influx. Since we have previously shown that AS is not associated with alterations in density and kinetics of ICaL (see Volk & Ehmke, 2002), a requirement of an increased Ca2+ influx is that the underlying L-type Ca2+ channels remain open for a longer period of time. To analyse the time course of the activation and inactivation gates during the course of the different APs, we used a recently published model of rat myocytes which is the first to include endo–epicardial differences and hence allows one to address problems specific for epicardial myocytes (Pandit et al. 2001; for details of modelling see Methods). Figure 4 displays a computed epicardial AP (Fig. 4A), AP-induced Ca2+ current (Fig. 4B), cumulative AP-induced Ca2+ influx (Fig. 4C), activation gate (Fig. 4D), the product of all inactivation gates (Fig. 4E, calculated as the product of the three inactivation gates), the relative conductance (Fig. 4F, calculated as the product of activation and inactivation gates), and the resulting conductance–voltage and current–voltage relations (Fig. 4G). The dashed lines correspond to traces that were calculated under similar conditions except that Ito magnitude was reduced by 30%. The modelled epicardial AP displays a similar shape to the average AP recorded from sham-operated animals. Furthermore, a reduction of Ito by 30% induced a prolongation of the AP that was quantitatively comparable to the effect of AS on the epicardial AP, indicating that the reduction of Ito is sufficient to explain the AP prolongation in AS animals. The AP-induced Ca2+ current was of similar magnitude to that observed in epicardial myocytes of sham-operated rats, in which Ito was normal. Again, a 30% reduction of Ito produced similar alterations in the modelled AP-induced Ca2+ current as did AS to the recorded current. The total amount of Ca2+ influx increased by 50%, primarily as a result of an increase in duration of the current (Fig. 4B and G), whereas the peak of the AP-induced Ca2+ current was marginally reduced. In a previous study we showed that, in addition to a 30% reduction in Ito, AS lead to a 25% reduction in IK1 (Volk et al. 2001). To estimate the potential contribution of a reduction in IK1 to the observed increase in APD and QCa, we also calculated the effect of a reduction in IK1 alone on AP and QCa. APD90 was only marginally increased, by 9%, while the increase in QCa was negligible, at 0.6%. A reduction of IK1 by 25% in addition to an already reduced Ito of 30% produced similar results, APD90 and QCa were only marginally affected (–8.7 and +0.7%, respectively, data not shown).
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Discussion |
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Methodological limitations
This study was designed to investigate the effect of cardiac hypertrophy induced by AS on regional differences of the AP-induced Ca2+ current in the left ventricle. Since cardiac hypertrophy can also alter intracellular Ca2+ handling and thus inactivation of L-type Ca2+ channels by Ca2+ (Ahmmed et al. 2000), intracellular Ca2+ was buffered with EGTA to abolish any influence of Ca2+ originating from the SR. Buffering of internal Ca2+ also prevents the activation of Ca2+-dependent currents, such as the Ca2+-dependent chloride current (ICl(Ca)) during the AP clamp recordings (Zygmunt & Gibbons, 1992). However, with EGTA present in the pipette solution, inactivation of L-type Ca2+ channels by Ca2+ entering the cell through the channels remains intact (Sham, 1997), most likely because the Ca2+-binding site is located very close to the channel mouth (de-Leon et al. 1995; Peterson et al. 1999). A contribution of Ca2+ released from the SR to L-type Ca2+ channel inactivation can be excluded, since the SR cannot be refilled by the SR Ca2+ ATPases because of the buffering of intracellular Ca2+. Consequently, due to an absence of a contribution of the SR to the inactivation of ICaL under our experimental conditions, we may have slightly overestimated APD and hence the magnitude of QCa. However, AP prolongation induced by 10 mM EGTA is relatively mild when compared to faster chelators of Ca2+, such as BAPTA (Fauconnier et al. 2003). This is in agreement with the observation that AP-induced Ca2+ currents recorded in the presence of 10 mM EGTA in the pipette solution differ only slightly from those recorded in the absence of EGTA (Linz & Meyer, 1998).
Differences between endo- and epicardial myocytes
The differences in APD and the values we obtained for QCa in endo- and epicardial myocytes are similar to those we have previously observed in normal rats (Volk et al. 1999) and are in the range which has been described by others for rat ventricular myocytes without differentiation between endo- and epicardial myocytes (Bouchard et al. 1995; Terracciano & MacLeod, 1997). In contrast to the present results, a recent study detected a smaller Ca2+ influx in endo- compared to epicardial myocytes isolated from the canine left ventricle (Bányász et al. 2003). A possible explanation for this apparent contradiction is that the canine epicardial AP displays a pronounced spike-and-dome morphology, leading to reopening of L-type Ca2+ channels, which will facilitate an increased Ca2+ influx (Bányász et al. 2003). The consequences of endo–epicardial differences in QCa are not yet fully understood. The greater Ca2+ influx observed in endocardial myocytes of the rat left ventricle may be responsible for the larger diastolic and systolic Ca2+ transients (Figueredo et al. 1993) and the larger amplitude in contraction (Clark et al. 1993) observed in endocardial myocytes of the rat left ventricle. Whether this is also the case in the intact ventricle remains to be determined.
Effect of AS on AP-induced Ca2+ influx
In a previous study we have shown that an increase of APD induced by a pharmacological inhibition of Ito preferentially increases QCa in epicardial myocytes. We have therefore postulated that pathologies associated with a decrease in Ito might similarly affect QCa predominantly in epicardial myocytes (Volk et al. 1999). This, together with the observation that Ito is reduced by about 30% in epicardial myocytes isolated from AS rats (Volk et al. 2001), predicts that QCa should increase by a factor of two in epicardial myocytes of AS animals. Strikingly, this was indeed observed in the present study. This result underlines the important role played by Ito as a modulator of shape and duration of the ventricular AP and so of the AP-induced Ca2+ influx. Moreover, the present results indicate that a reduction of Ito is the most important cause of the AP prolongation in epicardial myocytes of rats subjected to AS, as we have suggested previously (Volk et al. 2001). This is further supported by the results obtained from the model calculation, which revealed that a reduction of IK1 by 25% in control myocytes or in myocytes in which Ito was reduced by 30% did not further alter APD or AP-induced Ca2+ influx to a significant extent. The absence of any influence of AS on Ca2+ influx in endocardial myocytes is in agreement with previous studies that also failed to detect an effect of cardiac hypertrophy on AP shape and duration in endocardial myocytes (Shipsey et al. 1997; Volk et al. 2001). A close association of Ito magnitudes and AP-induced Ca2+ influx has also been observed in regions of the heart other than the left ventricle, such as the septum or the right ventricular free wall (Kaprielian et al. 2002), as well as in myocytes in which Ito was decreased in response to myocardial infarction (Kaprielian et al. 1999). The control of APD, Ca2+ influx and Ca2+ transients by the magnitude of Ito therefore appears to be a general mechanism operating in the entire rat ventricle. Recent results suggest that the magnitude of Ito may not only alter APD and Ca2+ transients, but could even influence the development of cardiac hypertrophy by altering Ca2+ handling of the myocytes (Sah et al. 2002a; Zobel et al. 2002). Hence, in addition to regional electrical alterations that are associated with an increased risk of ventricular arrhythmia and sudden cardiac death (Zipes & Wellens, 1998; Elming et al. 1998), one could speculate that a selective reduction of Ito leads to mechanical inhomogeneities in the left ventricle, such as regional differences in myocyte hypertrophy.
In a previous study we demonstrated that AS affects neither the magnitude nor the kinetics of the L-type Ca2+ current (Volk & Ehmke, 2002). Hence, the mechanism underlying the increased AP-induced Ca2+ influx in epicardial myocytes of AS animals cannot be ascribed to changes in ICaL characteristics, but rather to changes in AP waveform. Other studies investigating the effect of myocardial infarction in rats (Kaprielian et al. 1999) or endo–epicardial differences in the dog (Bányász et al. 2003) came to the same conclusion.
The calculation of the effect of a 30% reduction of Ito on the epicardial AP waveform and the corresponding AP-induced Ca2+ influx in the model of Pandit et al. (2001) produced very similar results to those obtained experimentally. This further underlines the strong influence that a reduction of Ito alone can exert on the shape of the AP waveform and so the AP-induced Ca2+ influx. Furthermore, the model analysis of ICaL gating suggests two possible mechanisms that may contribute to the altered Ca2+ influx in hypertrophied myocytes. First, the increase in AP-induced Ca2+ influx in response to a reduction of Ito by 30% is facilitated by an increased duration of opening of the activation gate rather than a delayed inactivation. Second, the increase in Ca2+ influx is limited by a facilitated closing of the inactivation gates. Such a pronounced inactivation of ICaL is in agreement with a previous study revealing that, in rat cardiomyocytes, ICaL inactivates to a much greater extent compared to species with longer APs, such as the guinea-pig or the rabbit (Linz & Meyer, 2000). The strong limitation of the increase in AP-induced Ca2+ influx may serve as an important mechanism to prevent an excessive Ca2+ influx in pathophysiological conditions that are associated with an increase in APD.
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