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(Circulation Research. 1996;78:15-25.)
© 1996 American Heart Association, Inc.

Articles

Developmental Changes in Ionic Channel Activity in the Embryonic Murine Heart

M.P. Davies, R.H. An, P. Doevendans, S. Kubalak, K.R. Chien, R.S. Kass

From the Department of Physiology, University of Rochester (NY) School of Medicine and Dentistry (M.P.D., R.H.A., R.S.K.), and the Department of Medicine, School of Medicine, University of California, San Diego (P.D., S.K., K.R.C.).

Correspondence to R.S. Kass, PhD, Department of Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. E-mail rsks@uhura.cc.rochester.edu.

	Abstract

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Abstract We have isolated murine embryonic atrial and ventricular cells derived from timed-pregnant females at different periods and used patch-clamp procedures to investigate age- and chamber-specific expression of ionic channels in the developing fetal mouse. Our data indicate that L-type Ca²⁺ channels play a dominant role in excitation during early murine cardiac embryogenesis and that Na⁺ channel expression increases dramatically just before birth. K⁺ channel expression is particularly sensitive to changes during development. Neither atrial nor ventricular cells express a slowly activating component of delayed rectification (I_Ks) until just before birth, and inwardly rectifying channel activity, associated with determination of cellular resting potential, is not markedly apparent until late stages of embryogenesis. Instead, we find robust expression of the ATP-regulated K⁺ channel at early and late stages of embryonic development, which may indicate a novel functional role for this channel during morphogenesis of the heart. These results have important implications for the physiology and development of the murine cardiac conduction system and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in the myocardium of both wild-type and genetically modified mice.

Key Words: ion channels • heart • development • mouse

	Introduction

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The use of transgenic and gene-targeting techniques has begun to provide insight into the molecular basis of cardiac development and dysfunction. The overexpression or selective knockout of genes in the genome of experimental animals produces models that can exhibit inherited cardiac pathology.^{1 2 3} Examples of this approach are widespread and include genetically based models of hypertension, cardiac hypertrophy/failure, atherogenesis, and developmental defects.^{3 4 5 6} With recent evidence that at least two forms of an inherited cardiac arrhythmia, the long-QT syndrome, are due to mutations of one or more ion channels expressed in the heart,^{7 8} it is clear that models of such disorders will need to combine genetically engineered animal models with information about the relation between expression of individual ion channels at the mRNA and protein levels. Although progress is being made in other species,⁹ the techniques for developing gene-targeted animals have been restricted primarily to mice because of the availability of totipotent murine embryonic stem cells. Despite this research, surprisingly little is known regarding the cellular electrophysiology of adult and, especially, developing murine cardiomyocytes, and changes in cardiac development induced by genetic perturbations are crucial to understanding the genesis of the disease process.

It is well documented that important electrophysiological changes occur during the embryonic development of mammalian and avian hearts. The levels of expression and the biophysical and pharmacological properties of ion channels change during the course of development.¹⁰ However, there is relatively little information on the developmental expression of cardiac ion channels in the embryonic mouse. To date, only the putative message for the time-dependent I_Ks has been reported in embryonic murine cardiomyocytes.¹¹ This mRNA was also identified in neonatal cells, in which I_Ks activity was also measured.¹² Additional data for multiple ion channel currents are available for neonatal mice¹³ and cardiomyocytes differentiated from pluripotent mouse embryonic stem cells.¹⁴

The purpose of this study was to systematically characterize the electrophysiological properties of cardiomyocytes derived from midgestational (11 to 13 days postcoitum) and late-gestational (17 to 20 days postcoitum) murine embryos (total gestation is 21 days). We focus on changes in ion channel expression during embryonic development in atrial and ventricular cells. To our knowledge, this is the first study that describes the developmental, chamber-specific expression of cardiac ion channels in the embryonic mouse. Our results provide evidence that L-type Ca²⁺ channel activity, expressed robustly throughout this period of development, plays important roles in the early stages of development of the mammalian cardiac electrical system and further that important changes in K⁺ channel expression take place during murine embryogenesis that affect the physiology of the developing heart.

	Materials and Methods

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Embryonic Heart Cell Culture
Pregnant mice were killed by cervical dislocation, and the uterus was removed and placed in PBS. The uterus was rinsed once and opened longitudinally to expose the embryos still contained within the decidua. Embryos were dissected from the uterus as described in detail by Sturm and Tam.¹⁵ We used a previously described method developed to culture myocytes derived from pregnant female (ICR, an outbred strain) mice at either 11 to 13 days or 17 to 20 days postcoitum.² Briefly, after placing the embryos in ADS buffer containing (in mmol/L) 116 NaCl, 20 HEPES, 1.0 NaH₂PO₄, 5.5 glucose, 5 KCl, and 0.8 MgSO₄, pH 7.35, the hearts were removed from the embryo. The atria can be distinguished by localization and color and were separated from the heart by pulling both atria free with forceps. The tissue was placed in an Eppendorf tube and spun briefly to replace the solution with fresh ADS buffer containing 0.5 mg/mL collagenase type II (Worthington) and 1.0 mg/mL pancreatin (GIBCO). Digestion was allowed for 15 minutes at 37°C on a rotating Ferris wheel followed by gentle trituration of the tissue. Tubes were quick spun and the supernatant was transferred to Dulbecco's modified Eagles's medium supplemented with 10% horse serum and 5% fetal calf serum. If necessary these steps can be repeated once. The cells can be plated on gelatin- and laminin-treated glass coverslips (1% gelatin and 20 µg/mL laminin, Sigma Chemical Co) or directly onto plastic coverslips. Electrophysiological recordings were made 18 to 48 hours after plating. No differences in ionic current properties were detected over this period in culture.

Experimental results shown in this article were obtained using patch-clamp procedures in conventional whole-cell or patch configurations¹⁶ as well as in the perforated-patch configuration.¹⁶ Intracellular and extracellular solutions, designed to isolate K⁺, Ca²⁺, and Na⁺ channel currents, have been described previously.^{17 18} Patch pipettes (Clay Adams glass) were pulled to resistances of 2.5 to 5.0 M when filled with intracellular solutions. For perforated-patch recording, nystatin was dissolved in methanol at a concentration of 20 mg/mL and then added to the standard internal solution to yield a final concentration of 150 µg/mL. Both the nystatin stock solution and the nystatin-containing pipette solution were subjected to 5 to 10 minutes of ultrasonication before use. Capacity transients were monitored as a function of time after attaining a high-resistance seal with the surface membrane, and electrical access to the cell was judged by the time course of the capacity transient. Adequate access was usually reached within 10 minutes of seal formation. Cell capacitance was determined either by integration of the capacity transient caused by 5-mV voltage step or analog measurement with the patch-clamp amplifier.

Distinct pulse protocols were chosen for each channel studied. In general, negative (-80 or -90 mV) holding potentials were used for investigation of Na⁺ channel currents, T-type channel currents, and I_Kr. Tetrodotoxin (10 µmol/L, Sigma Chemical Co) was added to solutions in which Na⁺ channels were to be eliminated, but holding potentials had to be negative. Nisoldipine (200 nmol/L) was added to solutions in experiments where L-type Ca²⁺ channel activity needed to be eliminated. More depolarized holding potentials (-50 or -40 mV) were generally used for measurement of L-type Ca²⁺ channels, delayed K⁺ channels, and inwardly rectifying K⁺ channels. These more positive holding potentials resulted in the inactivation of rapidly activating/inactivating K⁺ channels. When other voltage-gated, noninactivating K⁺ channels were activated at more positive potentials, these currents, along with linear leak, were subtracted. Holding potentials are specified in figure legends for each experiment. In general, brief pulses were used to assay channel activity to maximize cell endurance. I_Ks was measured by using 2-second activating pulses, and thus activation curves shown are isochronal and not steady state curves. All pulse durations and waveforms are described in individual figure legends.

Data were collected, stored, and analyzed on IBM 486–compatible computers interfaced to Yale Mark IV amplifiers constructed in our laboratory or to an Axopatch 200A amplifier driven by PCLAMP software (Axon Instruments). Graphics and statistical data analysis were carried out by ORIGINsoftware (Microcal).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Excitatory Currents
L-Type Ca²⁺ Channels
Our first experiments were designed to test for the presence of Ca²⁺ channel activity in both atrial and ventricular cells of early-stage embryonic hearts. We found L-type Ca²⁺ channel activity in 44 of 45 cells tested and did not find differential expression in the atrium compared with the ventricle. Fig 1 shows representative traces of L-type channel currents recorded from early-stage ventricular cells. As is well established in adult cells, L-type channel currents are smaller in amplitude and inactivate more quickly when Ca²⁺ (Fig 1A) and not Ba²⁺ (Fig 1B) is the charge carrier. L-channel current density increases during fetal days 11 through 20 (Fig 1C). We tested for and found dihydropyridine sensitivity of L-type channels at this stage of development but surprisingly did not detect sensitivity to catecholamine stimulation (data not shown but presented in detail in a subsequent study). We also failed to detect T-type Ca²⁺ channel activity in these cells when holding potentials were changed to voltages as negative as -90 mV (Fig 2), which contrasts with expression of this channel in the neonatal murine heart.¹³

View larger version (19K): [in this window] [in a new window]   Figure 1. Expression of L-type Ca2+ channel activity in fetal murine ventricular cells. A and B, Currents elicited by a series of depolarizing 50-millisecond pulses from -80 mV to +60 mV, with +10 mV increments, in 5 mmol/L Ca2+ (A, calibration: 200 pA, 10 milliseconds) or Ba2+ (B, calibration: 500 pA, 10 milliseconds) as the charge carrier. Recordings A and B are from the same myocyte. C, Mean current-voltage relations for early-stage (days 11 through 13, ) and late-stage (days 17 through 19, ) ventricular cells. Peak inward current amplitude was measured, normalized to cell capacitance, and plotted against test potential. Values in C are mean±SEM, n=7.

View larger version (19K): [in this window] [in a new window]   Figure 2. Absence of T-type Ca2+ channel currents in fetal mouse ventricular and atrial cells. A and B, Currents were elicited by depolarizing pulses from holding potential (VH) of -50 mV (A) or -90 mV (B) (calibration: 5 pA/pF, 10 milliseconds). Low-threshold fast-activating and -inactivating T-type current was not seen while holding potential was set at -90 mV (B). C, Current densities were plotted against test potentials (, VH=-50 mV; , VH=-90 mV) (mean±SEM, n=8). No significant difference was observed in activation threshold potential and peak current potential.

Na⁺ Channels
Na⁺ channels were also expressed at this stage of development, with biophysical properties very similar to Na⁺ channels in neonatal and adult hearts. Fig 3 shows examples of families of Na⁺ channel currents as well as summary data from a large number of cells that show the voltage dependence of activation for early-stage (days 11 through 13) and later-stage (days 17 through 20) embryos. Na⁺ channel expression was the same in ventricular and atrial cells at this stage. Na⁺ channels were blocked by tetrodotoxin (10 µmol/L) and inactivated in the steady state at -40 mV, as areNa⁺ channels in the adult heart (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3. Na+ channel current density increases during murine embryogenesis. A and B, Current induced by a series of depolarizing pulses from a holding potential of -80 mV to test pulses between -80 and +40 mV (+10-mV increments) in a day-11 through -13 (A, calibration: 5pA/pF, 10 milliseconds) or a day-17 through -19 (B, calibration: 50pA/pF, 10 milliseconds) mouse fetal ventricular cell. C, Mean current-density/voltage relations for peak inward current vs test potential (mean±SEM, n=4) for early-stage () and late-stage () cells.

Developmental Changes in Na⁺ and Ca²⁺ Channel Expression Levels
Fig 4 summarizes changes in the densities of Na⁺ and L-type Ca²⁺ channel currents that occur during embryogenesis. Changes in cell size occur during days 11 through 20 and can be monitored by changes in total cell capacitance. Fig 4A summarizes changes in capacitance over this period, showing that there is a 36% increase in capacitance and thus total membrane surface. During this same period of development, we found a much greater relative increase in the density of Na⁺ channel currents compared with L-type Ca²⁺ channel currents. Fig 4A also illustrates this point graphically, showing a 4.5 times greater increase in Na⁺ compared with L-type Ca²⁺ current densities, and indicates that this change takes place over a very narrow window of time. As shown in Fig 4B, for days 11 through 13 we found that all cells had some excitatory current activity and 34 of 45 cells had both Na⁺ and Ca²⁺ channel activity. However, 10 of 45 cells tested had only L-type Ca²⁺ channel activity. In only 1 of 45 cells tested were Na⁺ channels detected in the absence of Ca²⁺ channels. By days 17 through 20, this trend was not found; all cells tested had both Na⁺ and Ca²⁺ channel activity.

View larger version (13K): [in this window] [in a new window]   Figure 4. A, Developmental changes in cell size and expression of excitatory Na+ and Ca2+ channel currents. Total cell capacitance and current densities were measured at days 13, 16, and 19 of gestation. Peak inward current was determined for each case from the full current-voltage relationship and then averaged for each channel type and age group. Plotted in A are peak Na+ () and Ca2+() current densities (left y axis) and total membrane capacitance () (right y axis) vs age of cells (mean±SEM, n=14). B, Age-dependent changes in expression of excitatory currents. The bar graph is a summary of frequency of expression of cells expressing only Na+ channel currents (INa) or Ca2+ channel currents (ICa) in early-stage and late-stage fetal mouse ventricular cells. Each bar represents the percentage of cells expressing the particular channel in early-stage (days 11 through 13) (open bars) and late-stage (days 17 through 19) (filled bars) fetal mouse. All early-stage cells expressed Na+ channels, Ca2+ channels, or both channel types. All late-stage cells expressed both channels; none expressed only one type of excitatory channel.

Repolarizing Currents
We next studied K⁺ channel currents using solutions designed to isolate these channels and found a rich diversity of channel activity that changed markedly during embryogenesis. Because of the diversity of K⁺ channel expression, we have divided K⁺ channels into three broad groups and discuss our results within this organizational structure. Our results are discussed in terms of tissue-dependent and developmental stage–dependent differences in channel expression.

Rapidly Activating Channels
Fig 5 illustrates activity of three distinct rapidly activating K⁺ channels expressed in murine embryonic (days 11 through 13) cells. Fig 5A shows channel activity characterized by rapid activation and inactivation kinetics. We observed two components of this current in our experiments, one insensitive to 4-AP and one blocked by 4-AP (Fig 6), similar to reported properties of I_to.¹³ The IC₅₀ for block by 4-AP was 64 1.15 µmol/L. Fig 5B, upper panel, shows currents that activate rapidly but inactivate slowly and are also sensitive to 4-AP (data not shown). Fig 5C shows current insensitive to 4-AP that activates rapidly but does not inactivate, even during test pulses as long as 250 milliseconds.

View larger version (28K): [in this window] [in a new window]   Figure 5. Murine embryonic cardiomyocytes express rapidly activating K+ channels. The top panels show three types of rapidly activating K+ current in response to a series of test pulses (-40 mV to +80 or +100 mV, +20-mV increments). Elicited currents were rapidly inactivating (A), slowly inactivating (B), or very rapidly activating but noninactivating (C) currents. Bar graphs in the bottom panels summarize the frequency and tissue dependence of expression of each type of K+ channel. The percentage of cells that expressed each particular channel is shown for early-stage (days 11 through 13) and late-stage (days 17 through 20) atrial and ventricular cells (n=5 to 24). Filled bars represent the expression of currents in atrial cells and open bars the expression of currents in ventricular cells. The very small expression of the slowly inactivating currents and the noninactivating currents in late-stage atrial cells (*) is due in part to the dominance of the rapidly inactivating current in these cells, which may mask expression of other channels. Tetrodotoxin (10 µmol/L), nisoldipine (500 nmol/L), and E-4031 (5 µmol/L) were present to block Na+ channel currents, Ca2+ channel currents, and IKr, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 6. Rapidly inactivating K+ current is blocked by 4-AP. A, B, and C, Recordings of families of rapidly inactivating currents in response to depolarization (250 milliseconds, -40 to +100 mV, 20-mV increments) from a holding potential of -80 mV in the absence (A, calibration: 500 pA, 100 milliseconds) and presence (B) of 5 mmol/L 4-AP. Drug-sensitive current is shown in C. D, Concentration dependence of 4-AP inhibition (n=3 to 4 for each concentration). Normalized peak current amplitude (I/Imax±SEM) is plotted as a function of the log concentration of 4-AP. The continuous curve is a Boltzmann relationship determined by the best fit to the experimental data. The IC50 for the block was 1.15 µmol/L.

As a group, the rapidly activating voltage-dependent channels dominate K⁺ channel activity measured during development from day 11 through day 20 in murine embryonic atrial and ventricular cells. We found channels that inactivate rapidly expressed in 12 of 24 early-stage and 5 of 5 late-stage atrial cells but in only 5 of 28 ventricular cells. This chamber-dependent distribution was independent of developmental stage of the embryo. On the other hand, 10 of 19 early-stage and 4 of 9 late-stage ventricular cells expressed only slowly inactivating K⁺ channel activity (Fig 5B), and 6 of 19 early-stage and 3 of 9 late-stage ventricular cells expressed only noninactivating (Fig 5C) K⁺ channel activity, again independent of embryonic age. A small number (6 of 24) of early-stage atrial cells expressed the latter K⁺ channel activity in the absence of rapidly inactivating channels (Fig 5C). However, because virtually every late-stage atrial cell expressed a large component of rapidly inactivating channel activity, estimation of expression levels of slowly inactivating or noninactivating channels was difficult without full drug dissection (Fig 6). The lower panels of Fig 5 summarize tissue- and age-related differences in expression of rapidly activating K⁺ channels, and the likely underestimation of late-stage expression of the types of channel activity seen in Fig 5B and 5C is noted.

Delayed Rectifier Channels
We tested for the presence of two components of delayed rectification (I_Kr and I_Ks) that have been reported previously in neonatal murine heart¹³ and found that expression of this type of channel was subject to marked changes during early embryogenesis.

I_Kr is the dominant delayed rectifier K⁺ channel current expressed during fetal development (days 11 though 20). In fetal cells, as in adult tissue, this component of delayed rectification can be identified by its sensitivity to E-4031 (Fig 7A and 7B) and its voltage and time dependencies (Fig 7C and 7D). At early stages, I_Kr is expressed predominantly in the atrium (26 of 54 cells), but by day 20, I_Kr expression is roughly equal in atrial (6 of 8) and ventricular (3 of 5) cells (Fig 8). Despite the increase in the number of cells expressing this channel during this period of development, the expression levels, when normalized to cell capacitance, remain the same (Fig 7C and 7D).

View larger version (21K): [in this window] [in a new window]   Figure 7. IKr is expressed in early- and late-stage atrial myocytes and is identified by its voltage dependence and sensitivity to E-4031. A and B, Families of currents measured in response to 2-second pulses (-30 to +20 mV, 20-mV increments) in an early-stage (A) and a late-stage (B) atrial cell before and after application of 5 µmol/L E-4031 (holding potential, -40 mV). C and D, IKr expression levels do not change during the course of development. Shown are plots of current amplitudes, normalized to cell capacitance (mean±SEM), vs test voltage for time-dependent current measured during 2-second pulses (C) and for tail currents recorded upon return to the holding potential (D) in early-stage () and late-stage () atrial cells. The current measured during the pulse exhibits inward rectification, and the tail currents show saturation. Because E-4031 abolishes both tail current and time-dependent pulse current in these cells, the drug-sensitive tail current is the same as the total tail current in these experiments. Vm indicates membrane voltage.

View larger version (38K): [in this window] [in a new window]   Figure 8. Summary of the frequency of expression of IKs, IKr, IK1, and IKACh in early-stage (days 11 through 13) and late-stage (days 17 through 20) atrial and ventricular cells. Each bar represents the percentage of cells expressing the particular K+ channel current in atrial cells (filled bars) and ventricular cells (hatched bars).

For early-stage cells, we found only 1 of 55 ventricular cells expressing a small component of noninactivating current at very positive potentials that resembles I_Ks. This current was much more pronounced in the ventricle by day 20, when 7 of 14 cells tested had clearly identifiable I_Ks activity (Figs 8 and 9). We failed to detect I_Ks in fetal atrial cells for either early or late stages of development. In fact, in fetal atrial cells E-4031–sensitive current is equal to total delayed rectifier current, supporting the view that in atrial cells this is the sole delayed rectifier channel present.

View larger version (26K): [in this window] [in a new window]   Figure 9. Expression of IKs in late-stage (days 17 through 20) ventricular cells. Top, Currents elicited in response to a series of 2-second pulses (-30 to +70 mV, +20-mV increments), followed by return to the holding potential (-40 mV) for 2 seconds, in a late-stage (day 17 through 20) ventricular cell (calibration: 20 pA, 1 second). Bottom, Isochronal (2-second) current-voltage relation. Current amplitudes, normalized to cell capacitance (mean±SEM, n=5), measured at the end of the depolarizing pulse are expressed as a function of voltage. The continuous curve is for illustrative purposes only. This type of channel activity was not observed in early-stage ventricular or either early- or late-stage atrial cells. Vm indicates membrane voltage.

Inward Rectifiers
Inward rectifier (background) channel activity, which could most readily be identified by measuring currents negative to the K⁺ equilibrium potential, was studied first and found in only 5 of 28 atrial and 5 of 19 ventricular midgestational (days 11 through 13) cells but in virtually every (9 of 9 atrial, 4 of 4 ventricular) late-stage (days 17 through 20) cell tested (Fig 8). As was the case for I_Kr, I_K1 density does not change in cells that expressed this current (Fig 10), but the frequency of expression increased fourfold. No difference was detected between I_K1 expression in atrial and ventricular cells. I_KACh was found, as expected, only in atrial cells, but it was limited to later-stage cells (Figs 8 and 11). Because this channel is identified only by agonist induction, its absence at early developmental stages is likely to be linked to embryonic expression of the muscarinic receptor, which in rats undergoes marked increases during the last few days of gestation.¹⁹

View larger version (14K): [in this window] [in a new window]   Figure 10. Density of IK1 does not change during murine embryogenesis. A, Current density (mean±SEM), measured at the end of 50-millisecond pulses plotted against voltage in early-stage (, n=5) and late-stage (, n=4) ventricular cells. B, The same relation measured in early-stage (, n=5) and late-stage (, n=5) atrial cells. No significant changes in current density were measured in either cell type. Pulses were applied from a -40-mV holding potential. Inward rectification was most readily identified from currents measured in response to pulses negative to the holding potential. In cases when other voltage-dependent K+ channels were activated positive to the holding potential, these currents, along with linear leak, were subtracted. Vm indicates membrane voltage.

View larger version (20K): [in this window] [in a new window]   Figure 11. IKACh measured in late-stage (days 17 through 20) atrial cells. Top, Currents in response to a series of 50-millisecond pulses (holding potential, -40 mV; test pulses, -110 to +20 mV; 10-mV increments) before and after application of 2 µmol/L carbachol in a late-stage atrial cell (calibration: 500 pA, 10 milliseconds). Bottom, Mean current amplitudes, normalized to cell capacitance, measured at the end of 50-millisecond pulses in the absence (, n=5) and presence (, n=5) and after washout (, n=3) of 2 µmol/L carbachol. Early-stage (days 11 through 13) atrial cells were insensitive to carbachol. Vm indicates membrane voltage.

High Expression Levels of the ATP-Regulated K⁺ Channel
Perhaps most surprisingly, we found that murine embryonic cells express very high levels of the ATP-regulated K⁺ channel. Tests for the absence or presence of this channel were carried out with excised inside-out patches. Single-channel activity was tested for modulation by cytosolic [ATP]. Fig 12 shows an example of ATP-regulated K⁺ channel activity in an early-stage ventricular cell as well as histograms of single-channel current amplitude in the absence and presence of 2 mmol/L ATP (cytosolic). Using this approach, ATP-regulated K⁺ channel activity was detected in 4 of 4 early-stage (days 11 through 13) ventricular patches and 5 of 6 late-stage (days 17 through 20) ventricular patches. We also observed channel activity in 2 of 2 early-stage atrial cells. Frequency of detection of the ATP-regulated K+ channel did not change between day 11 and day 20.

View larger version (24K): [in this window] [in a new window]   Figure 12. Evidence for IKATP in an early-stage (days 11 through 13) fetal mouse ventricular cell. Top, ATP dependence of single-channel activity recorded from an inside-out patch of an early-stage (days 11 through 13) ventricular cell. Cytoplasmic [ATP] was changed as indicated. Solution changes were as indicated by arrows. Recordings were made in symmetric K+ solution at a -60-mV holding potential, and thus channel openings (unitary conductance: 63 pS) are seen as negative current. Bottom, The all-points histograms of single-channel current amplitudes in ATP-free (A) and 2-mmol/L ATP–containing (B) solutions.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Despite the widespread interest in the mouse as a model for genetically manipulated cardiovascular disease, this is the first study to report the functional properties and expression levels of ionic currents in the murine embryonic heart. Thus, the results presented in this study can serve as a baseline for future investigations of transgenic mouse models as well as for studies of developmental changes in ion channel expression in the wild-type murine heart. We have demonstrated that ion channel expression in the developing mouse heart can be age and chamber specific; therefore, several of our observations have important implications for the physiology and neural modulation of the mouse heart during embryogenesis.

By 11 to 13 days postcoitum the peristaltic-like contractions of the primitive heart tube have given way to more sequential contractions of the atria and ventricles, indicating the initial development of some form of organized conduction system. Atrial and ventricular separation is complete by day 13 to day 15, and the heart is divided into four definable chambers. Thus, by this stage of development it is assumed that the functional significance of the heart becomes essential to the continuing development and survival of the embryo. This theory is supported by recent transgenic studies in which double allelic knockout of the retinoid X receptor_a gene leads to a functionally compromised heart⁶ and embryonic lethality between day 13.5 and day 15.5.^{5 20} Concomitant with these morphological characteristics, the interconnections of the conduction system in the mouse also undergo significant developmental changes. The sinoatrial and atrioventricular nodes have become established by day 13, and specific conduction tracts are subsequently laid down.²¹ Clearly, understanding the activity of ion channels during this developmental window is essential, since beginning at day 13, survival of the embryo is dependent on a properly functioning heart.

Excitatory Currents
L-type Ca²⁺ channels and Na⁺ channels appear to be well developed as early as day 11 to day 13 (Figs 1 through 4). We did not observe any chamber-specific expression of these channels, and the biophysical and pharmacological properties of the embryonic channels were very similar to those reported by others for neonatal and adult hearts.^{13 22 23 24} In addition, Ca²⁺-dependent inactivation is preserved in the embryonic heart, which suggests that the ₁ subunit is expressed at this stage in much the same way as in the adult heart.²⁵ The observation that only modest increases in L-type channel density were measured between early and late stages of embryonic development, compared with significant increases in Na⁺ channel density (Fig 4), may have important physiological relevance. In rat and chick hearts, Na⁺ channel density is reported to increase markedly during the course of development,^{26 27} whereas L-type channel density increases less dramatically in the rat,²⁸ and decreases in channel density have been observed in developing chick hearts.²⁹ Although 75% of our cells at early-stage development (days 11 through 13) expressed both excitatory currents, 20% of early-stage cells expressed Ca²⁺ currents alone, and only one cell was shown to express Na⁺ currents without Ca²⁺ current. Taken together, these data suggest that L-type Ca²⁺ channels and not Na⁺ channels may play a dominant role in excitation at very early stages of development (earlier than days 11 through 13) throughout the heart. This is particularly interesting in view of the developmental expression of K⁺ channel activity that is important in determining cell resting potentials (see below).

Repolarizing Currents
Rapidly Activating K⁺ Currents
We found that unlike excitatory channels, channels that contribute to cellular repolarization in developing mouse cardiomyocytes were subject to chamber-specific expression. The dominant repolarizing currents in cells derived from either atrial or ventricular tissue were rapidly activating K⁺ currents, and we observed three distinct types of these currents that were age and chamber specific (Fig 5).

The predominant K⁺ channel current in atrial cells was a rapidly inactivating current similar to I_to. This type of channel activity has also been reported previously as dominant in neonatal cardiomyocytes, adult ventricular cells, and embryonic stem cells.^{13 14 30 31 32} The voltage-dependent, 4-AP–sensitive component is similar to the cloned Shaker-type K⁺ channel Kv1.4³³ and the Shal-type K⁺ channel Kv4.2³⁴ expressed in Xenopus oocytes. In rat cardiac tissue, the message for Kv1.4 and Kv4.2 is present at embryonic day 14, whereas the message for Kv1.2 is not detectable until after birth³⁵ ; however, no comparable data are available for the embryonic mouse heart.

The slowly inactivating current, which was similar to the adult rat atrial delayed rectifier and the neonatal canine epicardial delayed rectifier,^{36 37} possessed biophysical properties similar to the expressed Shaker-type channels Kv1.1, Kv1.2, and Kv1.5^{38 39 40} and the Shab-type channel Kv2.1.⁴¹ These channels are all sensitive to 4-AP, and transcripts for each of these channels, especially Kv1.5, have been detected in the developing rat heart.³⁵ The apparent absence of expression of the slowly inactivating channel in late-stage atrial cells (Fig 5) is misleading. The predominance of the I_to-like current in these cells made it difficult to identify the slowly inactivating current without complete pharmacological dissection, as recently described in human atrial cells.⁴²

The rapidly activating, noninactivating current may represent the component of native I_to that is also insensitive to 4-AP. The novel ultrarapidly activating delayed rectifier current identified in human atrial cells is sensitive to 4-AP⁴³ ; consequently, the current we report is unlikely to be a murine isoform of this channel. The background K⁺ current, or plateau current, identified in guinea pig myocytes is a possible candidate.⁴⁴ This current activates very rapidly and does not inactivate. The activation threshold is similar to that observed in our cells (-40mV). However, the sensitivity of the plateau current to 4-AP has not been determined.

Delayed Rectifier Channel Activity
In addition to rapidly activating K⁺ channels, we observed expression of both components (I_Kr and I_Ks) of delayed rectification that can be distinguished by slower activation and deactivation kinetics. Expression of these channels was also dependent upon age and tissue. E-4031–sensitive I_Kr was a dominant current in early-stage atrial cells. At this stage of development, I_Kr expression in ventricular cells was significantly lower than in atrial cells; however, in later-stage cardiomyocytes no chamber-specific expression was evident. The biophysical properties of I_Kr were very similar to I_Kr in adult guinea pig cells,⁴⁵ neonatal cardiomyocytes,¹³ atrial tumor cells derived from adult transgenic mice,⁴⁶ and human atrial cells.⁴⁷

Expression of I_Ks was less frequent than that of I_Kr. I_Ks was observed only in later-stage ventricular cells, with no expression detectable in atrial cells. These results were surprising, since I_Ks has been recorded in neonatal cells^{11 12 13} and the minK (or I_sK) message that is linked to this current¹⁸ has been detected in murine day-15 embryonic cells.¹¹ Because the expression of I_Ks in the murine embryonic heart is both tissue and age related, this preparation has the potential to provide a pivotal role in unraveling the relation between message for and expression of this delayed K⁺ channel in the heart.

Inward Rectifiers
The expression of I_K1 also undergoes significant changes during murine cardiac development. I_K1 expression increased fourfold between early and late stages in both atrial and ventricular cells, although no change in current density was observed. It is known that neonatal cardiac myocytes express an inward rectifier¹³ ; however, these are the first data on developmental changes of embryonic murine cardiac I_K1. I_K1 undergoes similar developmental changes in rat, rabbit, and chick hearts.^{48 49 50}

A Developmental Role for I_KATP?
We found I_KATP activity in virtually every patch of membrane for which its presence was assayed. The appearance of this channel did not depend on the presence of either I_K1 or I_KACh activity (Fig 12). Thus, the embryonic murine heart may serve as a powerful preparation in which to sort out the relation between the message for the channel protein that underlies I_KATP and the message for other inwardly rectifying K⁺ currents. Recent reports^{51 52} have suggested that the molecular identity of this channel may be more complicated than originally thought,⁵³ and a preparation with minimal inwardly rectifying K⁺ channel activity will be extremely useful in identifying a message with expressed channel activity.

Finally, the high level of expression of the ATP-regulated K⁺ channel in the embryonic murine heart naturally raises questions about possible functional roles of this channel during development of the heart. Because we found very low expression levels of inwardly rectifying K⁺ channels (Fig 8) in both chambers of early-stage murine embryonic heart cells, determination of cellular resting potential must depend on other ionic pathways, and certainly the ATP-regulated K⁺ channel becomes an attractive candidate pathway to consider in view of its relatively high expression level during early stages of embryogenesis. Future studies will be directed at determining whether or not biophysical properties of this channel, such as pH dependence and ATP dependence of channel open probability, differ in adult and embryonic cells and whether or not cellular resting potential itself plays a role in the rapid change in expression of Na⁺ compared with Ca²⁺ channel activity that we measured during murine embryogenesis.

Conclusion
In summary, we have investigated the ionic currents in different embryonic stages of the mouse heart and found marked chamber- and age-related differences in ion channel expression. Our results indicate a dominant role of L-type Ca²⁺ channels in early stages of murine embryogenesis, important changes in relative expression of K⁺ channel activity, and a putative novel role of the ATP-regulated K⁺channel during early stages of development of the mouse heart. These data should prove useful both for an understanding of the development of the electrical system of the mammalian heart and as a baseline against which changes in ion channel expression induced by models of cardiovascular disease generated by genetically engineered mice may be compared.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine IK1 = inwardly rectifying background K+ current IKACh = acetylcholine-activated K+ current IKATP = ATP-regulated K+ current IKr = rapidly activating delayed rectifier K+ current IKs = slowly activating delayed rectifier K+ current Ito = transient outward current Kv = voltage-gated K+ channel

Received June 15, 1995; accepted September 28, 1995.

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