Abstract Developmental changes in the transient outward K+ current (Ito) in mouse ventricular myocytes were assessed by the whole-cell patch-clamp technique. The density of Ito in mouse ventricular myocytes was significantly increased from the day-1 neonate to the adult. At +50 mV, the density of Ito was 3±1 pA/pF in the day-1 neonate, 15±3 pA/pF in the day-14 neonate, and 19±4 pA/pF in the adult (P<.01). Unlike other species, the rate of Ito inactivation significantly slowed in mouse ventricular cells during development. Moreover, the time courses of inactivation and recovery from inactivation of Ito were well described by a monoexponential function in day-1 neonatal cells, whereas they were best fitted by a biexponential function in day-14 neonatal and adult cells. The characteristics of steady state inactivation were also significantly different in day-1 neonatal cells (half-inactivation potential [Vh]=−66±4 mV, slope factor [k]=12±2 mV), in day-14 neonatal cells (Vh=−40±3 mV, k=13±1 mV), and in adult cells (Vh=−34±4 mV, k=6±1 mV). Microelectrode studies revealed that action potential duration progressively decreased in mouse ventricles during normal postnatal development. In addition, 4-aminopyridine (1 mmol/L) prolonged action potential duration more in adult than in neonatal mouse ventricles, suggesting that the developmental increase in the density of Ito contributes to the age-related shortening of action potential duration in mouse ventricles. In conclusion, Ito in adult mouse ventricular myocytes exhibits a higher density, slower inactivation kinetics, and a relatively more positive half-inactivation potential. All these characteristics result in Ito being a physiologically more important repolarizing K+ current in adult than in neonatal mouse hearts.
The voltage-dependent Ca2+-independent Ito has been recorded in mammalian cardiac myocytes from many species.1 2 3 4 5 Because of its rapid activation kinetics, Ito is responsible for the rapid initial phase of action potential repolarization. Recent studies have shown that in addition to its important role in electrophysiological heterogeneity in the heart,4 6 7 8 expression of Ito is also profoundly species and age dependent.9 10 11 12 13 Furthermore, age-related changes in cardiac action potential configuration have been postulated to be associated with developmental alteration in Ito in many species.10 11 However, only limited studies have focused on developmental expression of Ito and its functional role in the mouse heart. Davies et al14 studied the expression of Ito in mouse cardiac myocytes only during intrauterine development. Since the type and the density of other K+ currents are remarkably altered in the mouse ventricle after birth,15 the purpose of this investigation was to study the expression of Ito in the mouse ventricle during normal postnatal development. In the present study, we show that the density of Ito increases significantly and that its inactivation properties are profoundly altered in mouse ventricular myocytes during normal postnatal development. The results of the present study provide prerequisite knowledge for studying or designing transgenic mice with altered K+ channel expression in the heart. These results are also very useful for the understanding of K+ channel regulation in the mouse heart during normal development.
Materials and Methods
Neonatal day-1, neonatal day-14, and adult CD-1 mice were used for the present study. The mice were handled in accordance with our institutional guidelines for animal use in research.
Single Ventricular Myocyte Isolation
Single ventricular myocytes from day-1 neonatal mice were isolated and cultured by a previously described method,16 and single ventricular myocytes from day-14 neonatal and adult mice were isolated by a previously described Langendorff perfusion technique.15
Whole-Cell Patch-Clamp Recording
Macroscopic K+ currents were recorded by the whole-cell patch-clamp method with an Axopatch 200 amplifier (Axon Instruments). Electrodes had tip resistances of 1 to 4 MΩ for adult, day-14, and day-1 neonatal myocytes when filled with an internal solution containing (mmol/L) potassium aspartate 110, MgCl2 4, K2ATP 4.2, CaCl2 1, NaCl 8, HEPES 5, and EGTA 10, pH 7.2 adjusted with KOH. A liquid junction potential of ≈10 mV (pipette negative) was corrected electrically. Mouse ventricular myocytes were superfused at 2 mL/min at room temperature (22°C) with a HEPES-buffered Tyrode’s solution containing (mmol/L) NaCl 140, KCl 4, MgCl2 1, CaCl2 1, glucose 5.5, and HEPES 10, pH 7.4 adjusted with NaOH. L-Type Ca2+ current was blocked by CdCl2 (0.3 mmol/L). For action potential recordings, mouse ventricular myocytes were perfused with normal Tyrode’s solution in the absence of Ca2+ channel blocker. The cell capacitance and series resistance were measured and calculated from the uncompensated capacity current transients elicited by a 10-mV hyperpolarizing voltage step from a holding potential of −80 mV. Series resistance was checked regularly to ensure no variations with time. If the series resistance increased during the course of a recording, the data were discarded.
Action potentials from single ventricular cells were recorded at room temperature in the current-clamp mode of the whole-cell patch clamp. The membrane currents and action potentials were monitored on a storage oscilloscope, digitized, and stored on an IBM AT computer. Data acquisition was performed using a TL-1 DMA interface and pCLAMP software (Axon Instruments).
In addition, developmental changes of the action potential configuration in mouse ventricles were also assessed by a conventional microelectrode technique.15 Briefly, right ventricular myocardium was isolated from day-1 neonatal, day-14 neonatal, and adult mouse hearts. The preparations were superfused with an oxygenated Tyrode’s solution at 37°C. The myocardial preparations were constantly paced at 2 Hz, and action potentials were recorded before and after exposure to an Ito channel blocker, 4-AP.
Whole-cell patch-clamp data were analyzed using CLAMPFIT software and plotted using the Fig P graphic software (Biosoft, Cambridge, NJ). The action potential configurations recorded from microelectrode experiments were stored, processed, and analyzed using CELLSOFT (D. Bergman, University of Calgary [Canada]). All averaged and normalized data were presented as mean±SE unless otherwise indicated. Statistical significance among groups was determined using one-way ANOVA. A value of P<.05 was considered significantly different. To define the difference between the subgroups compared within ANOVA, such as the day-1 neonatal group versus the adult group, Dunnett’s multiple-range test was used.
Age-Related Changes in Morphology of Mouse Ventricular Myocytes With Light Microscopy
Fig 1⇓ shows phase-contrast photomicrographs of mouse ventricular myocytes taken on a Zeiss Axiovert inverted microscope equipped with a Contax SLR camera. The appearance of mouse ventricular myocytes changes remarkably during development. One day after birth, mouse ventricular myocytes were spherical in appearance without visible striations (Fig 1A⇓). After short-term cell culture (24 hours), day-1 neonatal cells flattened and spread somewhat (Fig 1B⇓). Two weeks after birth, mouse ventricular myocytes were spindle-shaped, and most cells had considerably less prominent striations than adult cells (Fig 1C⇓). In general, day-14 neonatal mouse ventricular myocytes were less than half of the width of adult mouse ventricular myocytes. Adult mouse ventricular cells were rod-shaped and exhibited clear cross striations (Fig 1D⇓).
As shown in Fig 1⇑, in addition to changes in the shape of mouse ventricular myocytes, changes in the size of mouse ventricular cells also occur during development. The average capacitance of mouse ventricular myocytes was 24±2 pF for day-1 neonatal mice, which significantly increased to 75±5 pF for day-14 neonatal mice and 136±9 pF for adult mice (P<.01).
Current-Voltage Relationships and Density
Fig 2⇓ illustrates the representative superimposed outward currents elicited by the protocol shown in the inset. The amplitude of the peak outward currents significantly increased from day-1 neonatal (Fig 2A⇓) to day-14 neonatal (Fig 2B⇓) and adult (Fig 2C⇓) mouse ventricular myocytes. The inactivation process of the current was incomplete in all three age groups, suggesting that the peak outward current consists of at least two components. Thus, the increase in the peak outward current may be due to an increase in Ito only, an increase in Isus only, or the combination of both. Therefore, the amplitudes of both peak current and Isus were measured, and then Ito was obtained by subtracting Isus from the peak outward current.
The current-voltage relationships of Ito and Isus from each age group are illustrated in Fig 3⇓. As shown in Fig 3A⇓, activation of Ito was voltage dependent in all three age groups. The density of Ito normalized to cell capacitance significantly increased in mouse ventricular myocytes during development (Fig 3A⇓). At +50 mV, the density of Ito increased significantly from 3±1 pA/pF in day-1 to 15±3 pA/pF in day-14 neonatal mouse ventricular myocytes (P<.01). In adult mouse ventricular myocytes, Ito further increased to 19±4 pA/pF. An increase in the density of Isus was also significant but less dramatic (Fig 3B⇓). Therefore, the developmental increase in the peak outward currents is mainly due to an increase in Ito.
Inactivation Kinetics of Ito
The inactivation rate of Ito in mouse ventricular cells was obviously altered during postnatal development (Fig 4⇓). The current in day-1 neonatal ventricular myocytes inactivated rapidly and reached a steady state within 250 milliseconds. In contrast, Ito in day-14 neonatal and adult mouse ventricular myocytes inactivated slowly and did not reach the steady state at the end of a 1000-millisecond depolarization pulse.
The inactivation time constants of Ito in day-1 neonatal, day-14 neonatal, and adult mouse ventricular myocytes were determined by an exponential fitting program in CLAMPFIT software. The inactivation kinetics of Ito in day-1 neonatal mouse ventricular myocytes were well described by a monoexponential function, suggesting that only one inactivation component contributes to the Ito at this age stage (Fig 4A⇑). However, the inactivation time course of Ito in day-14 neonatal mouse ventricular myocytes was not well described by a monoexponential equation, suggesting that there is more than one inactivation component in day-14 neonatal and adult mouse ventricular myocytes (Fig 4B⇑). The inactivation time course of Ito in adult mouse ventricular cells did not fit to a monoexponential function either, whereas the data from both neonatal day-14 and adult myocytes were very well fitted with a biexponential function. The inactivation time constants of Ito obtained from each of the age groups were averaged and displayed in Fig 4C⇑ and 4D⇑. Both fast and slow inactivation time constants of Ito were voltage independent at test potentials from +10 mV to +50 mV.
Steady State Inactivation of Ito
A two-pulse protocol was used to assess the voltage dependence of steady state inactivation of Ito. The prepulses were used to depolarize the cell to different membrane voltages ranging from −100 to 0 mV for 5 seconds. Each prepulse was followed by a single test pulse, which depolarized the cell to +50 mV. Representative current traces elicited by this double-pulse protocol from day-1 neonatal (Fig 5A⇓), day-14 neonatal (Fig 5C⇓), and adult (Fig 5E⇓) mouse ventricular myocytes are shown. The peak currents evoked from the prepulses of −90 to 0 mV were measured and normalized to the value obtained from a prepulse of −100 mV. The amplitude of the peak currents was reduced substantially when a prepulse progressively depolarized the cell membrane from −100 to 0 mV, suggesting that the number of available channels was decreased with membrane depolarization. In Fig 5⇓, panels B, D, and F are plots of the normalized data obtained from day-1 neonatal, day-14 neonatal, and adult mouse ventricular myocytes, respectively, as a function of prepulse voltage. The curves through each data point represent the best fit by the Boltzmann equation. The mean values of Vh and k obtained by the Boltzmann equation for day-1 neonates were −66±4 and 12±2 mV, respectively (n=6). Although the k value of the steady state inactivation curve was unchanged in the day-14 neonate (13±1 mV), its Vh was significantly shifted to −40±3 mV (P<.05). Adult mouse ventricular myocytes underwent steady state inactivation at a much more positive potential, with a Vh value of −34±4 mV (P<.05) and a steeper k value of 6±1 mV (P<.05, n=5). These results indicate that in day-1 neonatal mouse ventricular myocytes, Ito undergoes substantially steady state inactivation at potentials close to the resting membrane potential. For example, at −66 mV, at least 50% of the available channels have been inactivated in day-1 neonatal mouse ventricular myocytes. In contrast, at −66 mV, almost 100% of the channels are still available in adult mouse ventricular myocytes.
Recovery From Inactivation
A double-pulse protocol was used to measure recovery of Ito from inactivation. The first pulse depolarized the membrane from −80 to +50 mV, which was followed by a second pulse from −80 to +50 mV at variable interpulse intervals to determine the fraction of available Ito at a given recovery time. The representative examples of such recordings from each age group are shown in Fig 6A⇓ (day 1), 6C (day 14), and 6E (adult). The recovery time course of Ito from inactivation was best fit by a monoexponential equation in day-1 neonatal cells with a time constant of 133 milliseconds (Fig 6B⇓). However, a biexponential fit was required to adequately describe the recovery time course of Ito in day-14 neonatal and adult myocytes (Fig 6D⇓ and 6F⇓, respectively). The fast recovery time constants were 26 milliseconds for day-14 neonatal cells and 41 milliseconds in adult cells. The slow recovery time constants were 900 milliseconds for day-14 neonatal cells and 1620 milliseconds for adult cells. In order to determine if the fast and slow components of inactivation recover independently, the magnitudes of the peak current and the fast and slow components derived from biexponential fits were measured for the different interpulse intervals. For example, when the interpulse interval was 275 milliseconds the fast component had recovered completely, whereas the magnitude of the slow component had recovered to only 60% of the control pulse in 14-day-old neonatal myocytes. The finding that complete recovery of the fast component occurred independent of the complete recovery of the slow component suggests that the fast and slow components function independently. Further molecular biological studies are required to confirm the working hypothesis that different gene products underlie the different components of Ito.
Effects of 4-AP on Ito in Mouse Ventricular Myocytes
4-AP is widely used as a blocker of Ito.17 To examine effects of 4-AP on Ito in day-1 neonatal, day-14 neonatal, and adult mouse ventricular myocytes, a single depolarization pulse was applied to mouse ventricular myocytes from −80 to +50 mV for 1000 or 3500 milliseconds. The current elicited by this protocol was recorded drug free and after application of 4-AP (1 mmol/L). As shown in Fig 7⇓, 4⇑-AP profoundly decreased Ito in all three age groups. The inhibition effect of 4-AP on Ito could be reversed by washout with normal Tyrode’s solution.
Relations Between Developmental Changes in Ito and Action Potential Configuration
The higher density, slower inactivation rate, and relatively more positive Vh values of Ito in adult mouse ventricular myocytes led to the hypothesis that these developmental changes in Ito contribute to the age-related shortening of cardiac action potential in mouse ventricles during development. To test this hypothesis, Ito and the action potential were recorded in the same cell before and after application of 4-AP (2 mmol/L). When Ito was inhibited by 4-AP, 4-AP also significantly prolonged the action potential duration in adult mouse ventricular cells (Fig 8A⇓ and 8B⇓). In some adult cells, after application of 4-AP, the cell failed to repolarize, and early afterdepolarizations occurred (Fig 8C⇓). The effects of 4-AP on action potential duration in mouse ventricles were also assessed by using the conventional microelectrode technique under more physiological conditions. The right ventricular myocardium from day-1 neonatal, day-14 neonatal, and adult mice were perfused with normal Tyrode’s solution at 37°C and continuously paced at 2 Hz. As shown in Fig 9⇓, the action potentials in each age group were recorded before (open circle) and after (solid circle) the application of 1 mmol/L 4-AP. In the absence of 4-AP, the action potential recorded from day-1 neonatal myocardium displayed a relatively small amplitude of phase-1 repolarization, followed by a clear plateau. Action potential duration significantly shortened in day-14 neonatal and adult myocardium. Moreover, the initial phase of repolarization was considerably larger and more rapid without a discernible plateau phase in day-14 neonatal and adult mouse ventricular preparations. Although 4-AP prolonged the action potential duration in all three age groups, the percent prolongation of the action potential duration (relative to drug free) increased during development. In review, our data show that developmental changes in Ito are physiologically relevant and contribute to developmental changes in action potential configuration in the mouse ventricle.
The present study shows that developmental changes in the morphology of mouse ventricular myocytes are temporally associated with profound increases in the density of Ito and changes in its inactivation properties. The inactivation rate of Ito slows during normal postnatal development in mouse ventricular myocytes, which is substantially different from previously reported data in other species. In addition, Vh of steady state inactivation occurs at a relatively negative potential (−66 mV) in day-1 neonatal cells compared with a relatively more positive Vh in adult mouse ventricular myocytes (−34 mV). These observations indicate that Ito is more physiologically important in adult than in neonatal mouse ventricles.
Developmental Changes in Ito
Age-related changes in the expression of cardiac Ito have been investigated in many species, including rats, rabbits, dogs, and humans.1 2 3 4 5 Recently, Davies et al14 examined the age-related expression of Ito in mouse hearts during embryonic development. The present study extends this work by reporting a progressive increase in Ito expression in mouse ventricular myocytes from postnatal to adult development. The inactivation kinetics of Ito from neonatal to adult mouse ventricular myocytes display a different pattern of Ito inactivation from previous studies in other species.9 10 Generally, the inactivation time course in the neonatal heart is much slower than that in the mature heart. For example, Jeck and Boyden10 have shown a rapidly activating, slowly inactivating outward current in ≈23% of all neonatal canine epicardial cells. They reported that such a slowly inactivating current was never observed in adult myocytes. In contrast, the present study has shown that Ito inactivated more rapidly in newborn than in adult mouse ventricular myocytes. Nuss and Marban18 have also shown rapid inactivation of Ito in neonatal mouse ventricular myocytes. In addition, a developmental increase in Isus was noted in the present study. However, the magnitude of the age-related increase in Isus was less than that of Ito. Developmental increase in Isus was also reported in human atrial myocytes by Gross et al.13
Physiological Relevance of Developmental Increase of Ito in Mouse Ventricles
Although Ito is present in newborn mouse ventricular myocytes, at a membrane potential of −66 mV, at least half of the available Ito channels are unavailable because of steady state inactivation. In addition, the inactivation kinetics were faster in newborn than in adult myocytes. As a result, Ito is expected to be less available for action potential repolarization in early neonatal than in adult mouse ventricles. In contrast, Ito in adult mouse ventricular myocytes exhibits a larger amplitude and slow inactivation kinetics. At resting membrane potential, almost 100% of Ito channels would be expected to be available for action potential repolarization in adult mouse ventricular cells. From these voltage-clamp data, it was expected that Ito would be a more important repolarizing current in adult compared with early neonatal mouse ventricles. This prediction was supported by our microelectrode studies. The extent of action potential prolongation by 4-AP was significantly greater in adult than in day-1 neonatal mouse ventricular myocardium.
Adult Myocytes: Comparison With Previous Findings of Ito
A voltage-dependent 4-AP–sensitive Ito in adult mouse ventricular myocytes was first described by Benndorf and Nilius.2 In the present study, we show a similar current in adult mouse ventricular myocytes; however, differences also exist. First, Ito was elicited from different holding potentials: −50 mV in the study of Benndorf and Nilius versus −80 mV in the present study. Second, the duration of the depolarization pulses in the study of Benndorf and Nilius was only 50 milliseconds. Given such a short depolarizing pulse, the inactivation of Ito in adult mouse ventricular myocytes would be far from complete. Third, the experiments were carried out at different temperatures. All these factors may result in the different inactivation kinetics of Ito in adult mouse ventricular cells between the two studies.
The characteristics of Ito inactivation in adult mouse ventricular myocytes are different from those in other species.1 4 8 19 Typically, Ito activates and inactivates rapidly in adult ventricular myocytes. In addition, the inactivation time course of Ito is best fit by a single exponential function in ventricular myocytes isolated from rats.19 In contrast, the inactivation kinetics of Ito in adult mouse ventricular myocytes were much slower and were composed of two inactivation components: a fast inactivation component (69±7 milliseconds at +50 mV) and slow inactivation component (903±66 milliseconds at +50 mV). Two inactivation components of Ito were reported in rat atrial but not ventricular myocytes.20 Similar to that found in rat atrial cells, the time course of the recovery of the fast inactivation component in mouse ventricular cells (day 14 and adult) is independent of the recovery of the slow component. This result suggests that the fast and slow components are functionally independent currents. The molecular biological studies are required to determine whether independent gene products underlie these two components.
In addition to the fast and slow inactivation components of Ito, a rapidly activating, noninactivating current, Isus, was also present in adult mouse ventricular myocytes. Thus, the total outward current recorded in adult mouse ventricular myocytes appears to be the sum of at least three components. The slow inactivation component is unlikely to be due to the Ca2+-activated K+ channel, because we have used CdCl2 in external perfusion and EGTA (10 mmol/L) in the internal solution to block ICa2+ and buffer intracellular Ca2+, respectively.
The present study has a number of limitations. The regional and transmural differences in Ito distribution during development were not assessed. The day-1 neonatal mouse heart is too thin to accurately isolate epicardial versus endocardial myocytes. Since the present study mainly focuses on time-dependent expression of K+ current in mouse hearts, a parallel design was used to sample the whole ventricles at the different periods of development.
In the present study, Ito was measured at room temperature rather than at a more physiological temperature. Therefore, the properties of Ito observed in the present study may differ from those recorded at 37°C. Most previously reported biophysical studies of Ito were also recorded at room temperature, probably because Ito activates so rapidly and its amplitude is so large that potential problems of voltage-clamp control would be more likely at 37°C. Therefore, our data can be compared with those of previous investigations.
Different dispersion methods were used to isolate myocytes in day-1 neonates than were used in day-14 neonates or adult mice in the present study. A previous study has compared the characteristics of measured K+ currents in day-10 neonatal rat ventricular myocytes obtained by Langendorff perfusion versus the chunk method and found that the currents were qualitatively very similar.1 Even so, we cannot completely exclude the possibility that the differences in the properties of the currents in day-1 neonatal cells versus day-14 and adult cells may in part relate to the different isolation methods, although this seems unlikely.
With the advances in molecular techniques, transgenic mouse models will facilitate the mechanistic analysis of the range of K+ channel gene products that are expressed at different stages of development or by certain heart diseases. From this point of the view, the results obtained from present study not only illustrate the characteristics of K+ currents in mouse ventricular myocytes during normal postnatal development but also provide prerequisite knowledge for designing future studies of K+ currents in transgenic mice.
Selected Abbreviations and Acronyms
|I sus||=||sustained current|
|I to||=||transient outward K+ current|
We thank Drs W.R. Giles and R. Clark for their many helpful comments and suggestions and Dr R. Clark’s careful and critical reading of the manuscript. We also thank Dr Zhong-Ping Feng for taking the photographs of the mouse ventricular myocytes.
- Received December 3, 1996.
- Accepted April 22, 1997.
- © 1997 American Heart Association, Inc.
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