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1 Dipartimento di Biologia Evolutiva e Funzionale – Sezione Fisiologia, Università degli Studi di Parma, Viale G.P. Vsberti 11/A, 43100 Parma, Italy
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Abstract |
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5%. Ten per cent random changes in CL were detected as a 40% increase in CV of APD and tended to correlate with it (r
= 0.43). Block of ICaL depressed this correlation (r
= 0.23), whereas block of Ito significantly increased it (r
= 0.67); this was similar with linearly changing CL ramps (ranging ±10% and ±20% of 250 ms). We conclude that beat-to-beat APD variability, a major determinant of the propensity for development of arrhythmia in the heart, is present in isolated myocytes, where it is dependent on mean APD and pacing rate. Action potential duration shows a beat-to-beat positive correlation with preceding randomly/linearly changing CL, which can be pharmacologically modulated.
(Received 26 March 2007;
accepted after revision 14 June 2007; first published online 15 June 2007)
Corresponding author M. Zaniboni: Dipartimento di Biologia Evolutiva e Funzionale – Sezione Fisiologia, Università degli Studi di Parma, Viale G.P. Usberti 11A, 43100 Parma, Italy. Email: zaniboni{at}biol.unipr.it
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Introduction |
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In this work, we aimed to better define beat-to-beat APD variability at constant pacing rates and to study the cellular mechanism of APD adaptation to random and linear changes in pacing frequency, in the absence of autonomic and electrotonic effects, which have been shown to modulate these properties in the intact heart (Laurita et al. 1997; Ng et al. 2007). To this end, we first demonstrated how intrinsic beat-to-beat APD variability is dependent on both mean APD and pacing rate and how it limits the accuracy of classical estimates of repolarization properties. Then we developed a random-CL pacing protocol and tested the ability of each APD to track changes in the preceding CL during stimulations that mimicked the frequency and variability of rat heart rhythm. We finally showed how two ion channel blockers affected beat-to-beat APD variability during constant pacing and modulated the capability of the APD to track the preceding CL, during both randomly and linearly changing pacing trains.
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Methods |
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Single cells were enzymatically isolated from adult (6 months old, 400–500 g) male Wistar rat left ventricles. Each rat was anaesthetized with ether inhalation and killed by decapitation. The heart was rapidly removed, mounted on a Langendorff apparatus, and perfused at 37°C with the following sequence of solutions: Ca2+-free (control, no added calcium) Tyrode solution for 5 min to remove the blood, low-Ca2+ (0.1 mM) solution containing 1 mg ml–1 type 2 collagenase (Worthington, Lakewood, NJ, USA) and 0.1 mg ml–1 type XIV protease (Sigma Aldrich, Milan, Italy) for 20 min, and enzyme-free low-Ca2+ solution for 5 min. The left ventricle was then minced and shaken for 10 min in the low-Ca2+ solution. Myocytes were stored at room temperature in the control solution with 0.5 mM Ca2+. All experiments were performed within 2–8 h after isolation. The procedure was approved by the Veterinary Animal Care and Use Committee of the University of Parma and conformed to the National Ethical Guidelines (Italian Ministry of Health; D.L.vo 116, January 27, 1992).
Solutions
Isolation solution contained (mM): 126 NaCl, 22 dextrose, 5.0 MgCl2, 4.4 KCl, 20 taurine, 5 creatine, 5 sodium pyruvate, 1 NaH2PO4 and 24 Hepes (pH adjusted to 7.4 with NaOH). The solution was gassed with 100% O2. Normal Tyrode solution (NT) for bathing of cells during experiments contained the following (mM): 126 NaCl, 11 dextrose, 5.4 KCl, 1.0 MgCl2, 1.08 CaCl2 and 24 Hepes (pH adjusted to 7.4 with NaOH). In some experiments, NT was added with 5 mM of 4-aminopyridine (4-AP), a transient-outward potassium current (Ito) blocker, or with 10 µM of nifedipine (from a stock solution in DMSO), an L-type calcium current (ICaL) blocker. The pipette filling solution contained (mM): 113 KCl, 10 NaCl, 5.5 dextrose, 5 K2ATP, 0.5 MgCl2 and 10 Hepes (pH adjusted to 7.1 with KOH). A drop of storage solution containing cells was placed in the experimental chamber (2.5 ml) and superfused by gravity at a flow rate of about 2 ml min–1. The temperature of the solutions in the cell bath was 37°C.
Electrical recordings
Suction pipettes were made from borosilicate capillary tubing (Harvard Apparatus, Edenbridge, UK) and had a resistance, when filled, of 2–4 M
. Transmembrane potential (Vm) was recorded by means of an Axoclamp 2B amplifier (Axon Instruments, Union City, CA, USA), adopting the whole-cell configuration of the patch clamp technique. Transmembrane potential was digitized at a sampling frequency from 5 to 10 kHz with a 12-bit analog-to-digital converter (Digidata 1200 Series Interface, Axon Instruments). Before a cell was contacted with the pipette tip, the pipette potential was set to zero and the voltage drop across the pipette was compensated with the bridge balance. Action potentials were elicited by means of constant current injections (3 ms, 2–3 nA). The APD was calculated as the interval between the time of maximal upstroke velocity and the time when Vm reached –60 mV (APD–60 mV) and –20 mV (APD–20 mV). When not otherwise specified, mean, standard deviation (S.D.) and coefficient of variability (CV = 100 S.D./mean APD) of APD were calculated over 10 consecutive beats after the AP waveform reached a stable configuration. We used custom-made software written in Matlab (The Mathworks Inc., Natick, MA, USA) to measure these parameters in series of consecutive action potentials. An interpolation procedure over the digitized Vm traces allowed a better time resolution on the raw data acquired at 5 kHz. A program was also written in Matlab language in order to generate randomly changing and linearly changing trigger signals, the first being sequences of trigger signals with CL uniformly distributed within a given percentage (CLR) of the basic cycle length (BCL) of 250 ms, the second being sequences of saw-tooth varying CLs ranging between ±10% or ±20% of 250 ms. In addition, trigger signals for constant CL stimulations with pre-/postmature stimuli were first generated in Matlab and then used to drive Axoclamp 2B current injections during the experiment. An electronic analog stimulator (Crescent Electronics, Sandy, UT, USA) was used to trigger the amplifier in protocols where switches between two constant values of pacing frequency were applied. In order to minimize the effects of ion depletion and run-down of ionic currents inherent in the whole-cell technique, we checked for the invariance of AP waveform before and after each pacing train and avoided long stimulation protocols. All experiments were performed within 2 min after establishing the whole-cell configuration (individual stimulation protocols never exceeded 8 s), the only exceptions being a few low-frequency stimulations (1 and 0.2 Hz) and steady-state rate-dependency experiments, which never lasted more than 5 min. A previous study (Zaniboni et al. 2000) reported no difference, in terms of beat-to-beat APD variability, between the ruptured patch and the more technically demanding perforated patch; therefore, the latter was not adopted in this study.
Statistics
Results are presented as means ± S.E.M. Student's paired and unpaired t tests, one-way ANOVA, as well as Kolmogorov–Smirnov test to check for normality of distributions, were performed by means of SPSS software (SPSS, Chicago, IL, USA). Statistical significance was set at P < 0.05. Correlations between APD–60 mV and CL sequences were performed by means of the ‘CORRCOEF’ Matlab function on the two vectors.
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Results |
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Beat-to-beat variability of APD–60 mV was first measured in myocytes electrically stimulated at fixed pacing rates. Figure 1A shows beat-to-beat changes of APD–60 mV over several cycles of a cell paced at 1 Hz. Longer stimulations allowed analysis of frequency distribution in terms of Gaussian fits for series of APD–60 mV recorded in the same cell at three different pacing rates (Fig. 1B). All the distributions were normal (Kolmogorov–Smirnov test: P
= 0.77 for 5 Hz; P
= 0.95 for 1 Hz; and P
= 0.5 for 0.2 Hz), which suggests, although without proving it, a random origin for the process. Increasing the pacing rate brought about a significant (one-way ANOVA, P < 0.05) decrease in S.D.–60 mV (from 3.50 ms at 0.2 Hz to 1.31 ms at 5 Hz) and CV–60 mV (Fig. 1C). This effect was also studied in a larger group of cells (n
= 100) which were paced at only two frequencies and where a highly significant (P < 0.01) decrease in S.D.–60 mV (from 2.33 to 1.37 ms) and CV–60 mV (from 2.55 to 1.37%) was found when pacing rate was increased from 1 to 5 Hz. Figure 1D emphasizes the overall changes of APD–60 mV range and APD–60 mV variability with increasing pacing rate, as well as the increase in temporal dispersion with increasing intrinsic APD–60 mV. We note that rate-dependent shortening of APD was not a consistent finding in our pacing experiments, where in fact a minority of the entire cell population (2 out of the 9 cells of Fig. 1C), unspecifically isolated from the entire left ventricle, showed APD–60 mV rate-dependent prolongation but a similar rate dependency of CV–60 mV. The two groups of myocytes, unambiguously identified from the positive or negative slope of their APD–60 mV rate dependency, did not significantly differ in resting membrane potential (–75 mV), action potential amplitude (93 mV) and APD–60 mV (87 ms, measured at a BCL of 250 ms).
We further characterized beat-to-beat APD–60 mV variability at a BCL of 250 ms, which is comparable to the rat physiological sinus rhythm. Figure 2A shows APD–60 mV-dependent increase of S.D.–60 mV for cells perfused in NT, in 10 µM nifedipine or in 5 mM 4-AP. In control conditions, for example, shorter values of APD–60 mV (50 ms) ranged ±3 ms (±3S.D.), whereas longer values of APD–60 mV ranged up to ±9 ms. Nifedipine, as expected, shortened the entire APD–60 mV range, whereas 4-AP slightly prolonged it, none of the treatments producing a significant change in the S.D.–60 mV range for the entire cell population. Overall drug-induced differences in beat-to-beat APD–60 mV variability became significant when measured as S.D.–60 mV normalized to mean APD–60 mV (CV–60 mV), as shown in the frequency distributions of Fig. 2B. Nifedipine significantly increased the average CV–60 mV value, whereas 4-AP reduced the same parameter.
Action potential duration rate dependency and electrical restitution properties
In order to estimate how beat-to-beat APD variability will limit the detection of rate-dependent effects on cellular repolarization, we performed two classical stimulation protocols. Steady-state rate dependency of APD–60 mV was quantified for small (< ±20%) changes (
CL) in BCL. Cells were paced for 13 s at a BCL of 250 ms, which was suddenly switched to different constant values, allowing each time for the action potential to reach a new steady-state configuration (100 beats). It appears (Fig. 3A) that steady-state rate-dependent APD changes for |CL| = 4% fell very close to the average intrinsic beat-to-beat variability range of APD–60 mV (hatched horizontal region), and only higher CLs were likely to be detected in terms of stable changes in recovery time. In Fig. 3B we report results of experiments on electrical restitution where only the last 10 conditioning beats at a BCL of 250 ms and a pre-/postmature test beat were measured. Since the test stimulus is the equivalent of the first stimulus at a new pacing rate, and for homogeneity with Fig. 3A, we refer to it, on the abscissa, as a new CL value. Instantaneous AP sensitivity to sudden changes of CL appeared to be asymmetric, where sudden prolongations of CL were sensed more, in terms of APD–60 mV, than sudden shortenings. Even very small increases in CL of only 4% were in fact detected, on average, as measurable (larger than beat-to-beat variability) APD–60 mV prolongations, whereas only considerable decreases (> 12%) were detected as measurable APD–60 mV shortenings. A minority of cells (n
= 4 for the rate-dependency experiments and n
= 3 for the restitution experiments) showed rate-dependent APD–60 mV prolongation and a negative sloping restitution curve. The corresponding data, comparable in amplitude of changes but with opposite sign, are reported in Fig. 3C.
Beat-to-beat APD variability in random-frequency stimulations
Beat-to-beat variability of APD–60 mV (CV–60 mV) was measured in myocytes perfused in NT and electrically stimulated, initially at a BCL of 250 ms (CLR = 0%), and subsequently with randomly variable CL trains with progressively increased degree of randomness (CLR, see Methods; Fig. 4). The average CV–60 mV value measured at CLR = 0% (dashed horizontal line) did not change significantly until CLR reached 6%, where it started to increase with the increasing of CLR. Some of the cells underwent the same protocol after drug exposure (not shown except for the data at CLR = 0% reported in Fig. 2). Nifedipine significantly increased CV–60 mV at almost all values of CLR (+30% on average), whereas 4-AP decreased it (–24% on average).
Correlation between APD–60 mV and CL for random fluctuations of CL: a ‘random restitution’ curve
Figure 5 reports scatter graphs for several superimposed 10-beat random stimulations (CLR
= 10%) of APD–60 mV values versus preceding CL (we will call them briefly ‘random restitution curve’). It appears that the increased variability in APD–60 mV measured at CLR
= 10% in NT (Fig. 4) was not random, but tended to correlate positively with beat-to-beat changes in CL (average correlation coefficient, r
= 0.43; single statistically significant coefficients were 11 out of 21; slope = 0.04% ms–1). In other words, a beat-to-beat shortening of the preceding CL tended to make APD shorter, and vice versa for a beat-to-beat prolongation of CL (see also the time sequence in the insets of Fig. 5A, B and C). Random stimulations in NT with higher CLR (20%) reached higher correlation (r
= 0.71; single statistically significant coefficients were 10 out of 13; and slope = 0.06% ms–1, not shown). Nifedipine-induced shortening of APD tended to abolish this correlation (r
= 0.23; none of the single coefficients was statistically significant; slope = 0.03% ms–1), whereas 4-AP-induced prolongation brought it up to statistical significance (r
= 0.67; single statistically significant coefficients were 7 out of 9; slope = 0.06% ms–1; see Fig. 5B, C and D). Analogous behaviour was found when restitution was measured as APD–60 mV
versus preceding DI (NT, positive slope with r
= 0.48; nifedipine, r
= 0.22; 4-AP increased slope with r
= 0.62).
Correlation between APD–60 mV and CL for linearly changing CL
The property of nifedipine and 4-AP of, respectively, uncoupling and coupling APD–60 mV with the preceding CL was further demonstrated with saw-tooth pacing protocols ranging ±10 and ±20% of a BCL of 250 ms (Fig. 6). With the ±10% protocol, APD–60 mV usually tended to correlate positively with monotonically changing preceding CL (r = 0.45), nifedipine dramatically reduced this correlation (R = –0.04), whereas 4-AP significantly increased it (r = 0.80; Fig. 6A). Analogous results were obtained with ±20% ramps (Fig. 6B, C and D). A tendency for rate-dependent APD–60 mV prolongation in NT was found in only two myocytes of those subsequently perfused with 4-AP and two of those subsequently perfused with nifedipine, which were not included in the previous statistic. We note, however, that in those cases the exposure to the blockers also led to a significant positive correlation in the case of 4-AP (r = 0.70) and a to loss of correlation (R = –0.31) in the case of nifedipine.
Measurements of APD during early repolarization
All measurements reported in this study for APD–60 mV have also been performed for APD–20 mV. Briefly, at CLR = 0%, APD–20 mV always (in NT, nifedipine and 4-AP) correlated with APD–60 mV (r = 0.67 on average) over 10-beat sequences. CV–20 mV was much greater than CV–60 mV (twice on average) and prevented the detection of classical rate-dependent and restitution properties within a CL range of ±10%. No significant drug- or rate-induced difference was measurable at CV–20 mV, nor could any drug-induced difference be assessed in the APD–20 mV–CL correlation during randomly or linearly changing CL protocols.
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Discussion |
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Zaniboni et al. (2000) reported a CV of 2.3% for the beat-to-beat APD variability of guinea-pig ventricular myocytes paced at 0.5 Hz. This fits with the CV–60 mV versus CL relationship we illustrate in Fig. 1C for rat myocytes, although measurements in the present study have been performed in terms of APD–60 mV (see Methods) instead of APD at 90% of repolarization. Our additional attempt to measure temporal dispersion of APD in the early phase of repolarization leads us to conclude that the variability of APD–20 mV, although qualitatively reproducing the variability of APD–60 mV (they are correlated), is a less appropriate parameter to detect beat-to-beat repolarization changes in this cell type.
Also in accordance with the cited guinea-pig study, longer rat ventricular APDs are found to be more temporally dispersed. We show here, for the first time, that this relationship holds at different pacing rates (Fig. 1D) and for different pharmacological treatments (Fig. 2A), and we suggest in the Appendix an explanation for this effect, based on the resistive properties of the cell membrane during repolarization.
While the increase of heart rate does not induce detectable changes in the dispersion of endocardial monophasic APD in dogs and humans (Hirao et al. 1996; Zabel et al. 1997), it is found to significantly decrease the increased dispersion of APD in long QT syndrome patients with pathologically prolonged action potentials, supporting the efficacy of pacemaker therapy to decrease propensity for arrhythmia (Hirao et al. 1996). The present study shows, for the first time, that the rate dependency of APD variability, measured as either S.D.–60 mV or CV–60 mV (Fig. 1C and D), is intrinsically present in the isolated cardiomyocyte also in non-pathological conditions, in the absence of higher level controls (e.g. electrotonic interactions, autonomic modulation, spatial sequence of activation) which operate within the whole organ. Cellular beat-to-beat APD variability and its APD dependency and rate dependency are not likely to be critical within the tissue in normal conditions, where temporal dispersion is reduced by electrotonic interactions (Zaniboni et al. 2000). In contrast, junctional uncoupling can unmask intrinsic beat-to-beat variability of APD, leading to a substrate more prone to unidirectional block and re-entry (Lesh et al. 1989). It is in this context that the intrinsic APD dependency and rate dependency of beat-to-beat repolarization variability might contribute to the initiation of arrhythmic events, especially in those pathologies (e.g. heart failure), or in ageing, which simultaneously decrease intercellular coupling and sinus rhythm, while increasing APD (Janse, 2004; Severs et al. 2004).
Our further finding that intrinsic APD variability at a BCL of 250 ms increases only for random changes of CL greater than 4% (Fig. 4) and tends to correlate with them on a beat-to-beat basis (Fig. 5), has not been shown previously in isolated ventricular myocytes. A 50% rise of CV of APD, secondary to a rise in CV of CL (from 2.3 to 5%), has been reported by Rocchetti et al. (2000) in a different cell type (spontaneously beating sinoatrial myocytes) following acetylcholine application, while a correlation between the RT segment of ECG and the preceding CL, although for much larger random CL changes, has been found in dogs (Lux & Ershler, 2003). The range we chose for CL variability (2–10%, some experiments at 20%) during rate-varying stimulations is comparable with beat-to-beat oscillations of rat sinus rhythm, which are reported to be around 4% in normal resting conditions, and to vary physiologically between 2 and 8%, for example during and following episodes of social stress (Sgoifo et al. 1998); vagal recruitment can bring these values to even higher physiological levels (Schipke & Pelzer, 2001; Hautala et al. 2001).
Based on previous findings that showed a differential contribution of different ion channels to beat-to-beat APD variability (Zaniboni et al. 2000), we wanted to test the relative contribution of ICaL and Ito, major determinants of membrane repolarization and restitution properties of rat cardiomyocytes (Nanasi et al. 1996; Janvier et al. 1997), in mediating interbeat APD variability during constant pacing rate and in modulating APD–CL correlation during randomly/linearly changing pacing rate. Nifedipine and 4-AP, together with their known shortening/prolonging effect on ventricular AP, brought about an overall increase/decrease in beat-to-beat APD variability (Fig. 2) which cannot be derived from the argument in the Appendix and seems to be related, instead, to the different extent of interbeat stochastic changes in the amount of ICaL and Ito flowing during the course of an action potential. The picture is consistent with (1) an ICaL endowed with a stabilizing effect on repolarization which is achieved by coupling APD with preceding CL, most likely through residual voltage- and calcium-dependent inactivation and through intermediate steps of calcium handling, (2) an Ito acting as a source of random changes on AP trajectory, uncoupling APD from preceding CL. Therefore, nifedipine-induced APD shortening allows more time for diastolic recovery of both ion channels from inactivation and resetting of calcium handling mechanisms, unmasking beat-to-beat random changes of Ito at constant pacing rate (Fig. 2B) and uncoupling each APD from the preceding CL (loss of correlation), in both randomly and linearly changing pacing protocols (Figs 5 and 6). Conversely, 4-AP decreases the source of interbeat random changes of APD at constant pacing rate (Fig. 2B) and, by shortening the diastole, prevents complete removal of inactivation processes and resetting of calcium handling mechanisms, coupling each APD with the preceding CL during rate-varying stimulations (higher correlation; Figs 5 and 6). Both blockers, by uncoupling APD from the preceding beat and by reducing the ionic source of interbeat stochastic changes, respectively, overbalance the intrinsic APD dependency of APD variability. A direct effect of these blockers on intrinsic beat-to-beat variability of APD has not been shown previously and is of interest, given that one of the beneficial effects of anti-arrhythmic drugs is thought to be their ability to decrease temporal dispersion of repolarization in the heart (Kuo et al. 1983; Fynn et al. 2003). We note that the mechanism we propose for the involvement of ICaL and Ito in APD variability is also consistent with the rate dependency of CV. In fact, an increase of the pacing frequency from 0.5 to 3 Hz is reported to decrease Ito by more than 50% in rat ventricular myocytes (Pandit et al. 2001), meanwhile reducing ICaL by only 30% (McMorn et al. 1998). In other words, increasing the pacing rate will have a greater impact in reducing random changes of Ito during early repolarization, resulting in an overall decrease of CV–60 mV.
Premature beats frequently initiate ventricular arrhythmias in ischaemic conditions (Janvier et al. 1997), and electrical restitution provides a tool for predicting such events (Franz, 2003). Nifedipine has been reported to decrease, and 4-AP to increase, the slope of the restitution curve, both in single cells, although at non-physiological pacing rates (Nanasi et al. 1996; Janvier et al. 1997), and in tissue preparations, where the pharmacologically induced slope reduction has been proposed to prevent oscillations of APD which lead to development of VF (Riccio et al. 1999). Our work shows, for the first time, that this effect of nifedipine and 4-AP can be measured at the cellular level in physiologically dynamic pacing conditions, where the slope of the corresponding random restitution curve (Fig. 5A) is comparable with that of classical restitution within the same CL range. This could not be simply predicted from classical rate-dependent and restitution properties, since these are measured at the steady state of membrane excitability and calcium handling, which is never achieved in dynamic conditions. A random pacing approach to cellular restitution might therefore represent a suitable model for further exploration of the intrinsic cellular counterpart of the more complex dynamic restitution of the heart.
Limitations of the study
An intrinsic limitation with any pharmacological block of specific ion channels in current clamp experiments is that AP waveform undergoes changes which in turn modify the relative contribution of all the ionic currents involved in repolarization. The mechanism we propose for the 4-AP and nifedipine effect on APD variability, based on stochastic contribution of only two ionic currents, does not rule out the possible involvement of other currents secondarily affected by the block, although it coherently explains all the data presented. Simulations by means of mathematical models of the rat ventricular action potential (Pandit et al. 2001), including controlled sources of variability, would be of interest at this regard. Also, we did not investigate the possibility that other mechanisms, such as those involved in intracellular calcium dynamics, or other ion channels involved in electrical restitution (e.g. delayed rectifier K+ current (IK), Na+-Ca2+ exchange current (INaCa); Janvier et al. 1997) would play some role in APD variability at constant pacing or in APD–CL correlation during randomly and linearly changing pacing rates. Finally, the rat ventricle, although widely used as an experimental model for the study of electrical heterogeneity in normal and pathophysiological conditions (see references in the Introduction of the paper by Pandit et al. 2001), has a peculiar AP profile (lacking plateau) and rate-dependent properties (Shigematsu et al. 1997; Carmeliet, 2006), which call for caution in extrapolating our results to other mammals and, in particular, to humans. A complication arises from the expected finding, together with rate-dependent APD shortening myocytes, of rate-dependent APD prolonging myocytes, owing to spatial heterogeneity in repolarization properties and differences in intracellular buffering conditions (Watanabe et al. 1983; Shipsey et al. 1997; Shigematsu et al. 1997; Fauconnier et al. 2003). These latter cells (between 22 and 40% in our experiments), unambiguously identified from the negative slope of their restitution curve, did not, however, show differences in the rate dependency of their beat-to-beat APD–60 mV dispersion, in the absolute value of rate-dependent and classical restitution changes, or in the pharmacological response during linearly CL-varying stimulations.
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| (1) |
–40 mV, is characterized, in each waveform, by a fairly constant membrane slope conductance, whose average value we derived from linear fit to the curves over this region. Average Rm (inverse of conductance) increases with the associated APD (Fig. 7B), since the relationship is quasi-linear for APD–60 mV up to 90 ms and then diverges for longer waveforms. We note incidentally that Rm during early repolarization of AP1 is identical to that of the resting membrane (40 M, inverse slope of dashed line, Fig. 7B), which is in accordance with recently published DC estimates in the same cell type (Zaniboni et al. 2005). We suggest that the increase of early repolarization-Rm with increasing APD can be the source of the relationship between S.D.–60 mV and mean APD–60 mV of Fig. 2A. Accordingly, longer AP waveforms will have an early repolarization phase characterized by a higher Rm, whereas shorter APs will have a lower resistance. Even in the simplified assumption that each action potential waveform will experience the same random electrical disturbances (the product of beat-to-beat changes in membrane ionic current resulting from stochastic behaviour of the channels and the instantaneous value of Rm) during the time in which most of the repolarization ionic current flows (early repolarization), shorter APs will deviate their interbeat trajectory to a smaller extent (smaller S.D. of APD) than longer APs.
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References |
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) and 12 cells in 4-AP (
). Measurements in NT are pooled controls from the nifedipine and 4-AP experiments. Each point represents S.D.–60 mV of a 10-beat sequence versus the corresponding mean APD–60 mV for one cell paced at a BCL of 250 ms. Superimposed curves are parabolic fittings to emphasize the S.D. increase with APD graphically. Inset shows representative examples of the effect of ion channel blockers (dotted lines) on AP waveforms at a BCL of 250 ms, compared with controls (continuous lines). B, frequency distributions for all the 10-beat sequences shown in A; the total number of beats (TNB) was 130 for nifedipine, 250 for NT and 120 for 4-AP. In order to allow comparison between Gaussian fits, ordinates are normalized to 1 (#), and abscissa to mean APD–60 mV value over each 10-beat sequence. Inset shows statistics of CV–60 mV for the same data (*P < 0.05).
P = 0.06).
