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Circulation. 1996;93:168-177

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(Circulation. 1996;93:168-177.)
© 1996 American Heart Association, Inc.

Articles

Regional Differences in Current Density and Rate-Dependent Properties of the Transient Outward Current in Subepicardial and Subendocardial Myocytes of Human Left Ventricle

Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993, and published in abstract form in Circulation (1993;88[suppl I]:I-89).

Michael Näbauer, MD; Dirk J. Beuckelmann, MD; Peter Überfuhr, MD; Gerhard Steinbeck, MD

From the Departments of Medicine I (M.N., G.S.) and Cardiovascular Surgery (P.Ü.), University of Munich, and the Department of Medicine III, University of Cologne (D.J.B.), Cologne, Germany.

Correspondence to Dr Michael Näbauer, Medizinische Klinik I, Klinikum Großhadern, 81377 Munich, Germany.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background Recordings of outward currents in human ventricular myocytes revealed the presence of a large calcium-insensitive transient outward current. This current has been suggested to contribute significantly to regional electrophysiological heterogeneity in myocardial cells and tissue of several animal species and to cause electrical gradients across the ventricular wall.

Methods and Results The patch-clamp technique was used to record action potentials and outward currents in myocytes enzymatically isolated from thin subepicardial and subendocardial layers of human nonfailing and failing left ventricle. In all subepicardial cells studied, a calcium-insensitive transient outward current (Ito1) could be recorded with large density (10.6±1.08 pA/pF at 40 mV), whereas current density of Ito1 in subendocardial cells was fourfold smaller (2.63±0.31 pA/pF, P<.0001, nonfailing myocardium). In failing hearts, the density of Ito1 was significantly smaller in subepicardial cells (7.81±0.53 pA/pF, P=.012) but not different in subendocardial myocytes (2.01±0.23 pA/pF, P=.25). Rate-dependent reduction of peak Ito1 at a 2-Hz depolarization rate was minimal in subepicardial cells (to 92.3±1.9%), whereas peak Ito1 in subendocardial myocytes was almost suppressed at 2 Hz (reduction to 13.2±2.1%, P<.0001). The different rate-dependent reduction of the transient outward current was due to a much slower time course of recovery from inactivation in subendocardial cells. Kinetic data, including action potentials recorded at 35°C, allow assessment of the role of the transient outward current for electrical activity and transmural voltage gradients in human left ventricle.

Conclusions Marked regional differences in density and rate-dependent properties of the transient outward current exist in subendocardial and subepicardial layers in human left ventricular myocardium, causing transmural electrical gradients that are important for normal and pathological electrical behavior of the human heart. The difference in recovery rates of the transient outward current is a distinguishing feature between subepicardial and subendocardial myocytes.


Key Words: action potentials • electrophysiology • heart failure • ventricles


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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Electrical heterogeneity within the ventricular myocardium of the mammalian heart has been described in several animal species and is considered to be an important factor for the electrical properties of the heart.1 2 3 4 5 Regional electrical differences within different layers of the working myocardium were first recognized by use of action potential recordings in syncytial preparations.6 The similarity of action potential characteristics of syncytial preparations and isolated myocytes of identical regions of the myocardium has led to the conclusion that regional differences in electrical behavior are due to a large part to different intrinsic properties of the myocytes.1 The ionic basis for these intrinsic electrophysiological differences, however, is largely unknown. Recent data indicate that different densities of a 4-aminopyridine–sensitive, calcium-independent transient outward current (Ito1) contributes significantly to regional electrical heterogeneity within the left ventricular wall1 6 because current densities several times larger in subepicardial than in subendocardial myocytes have been found in dog, cat, and, to a lesser degree, rabbit left ventricle.4 7 8 In canine left ventricle, where regional electrical differences have been studied most extensively, the difference in density of the transient outward current was approximately fivefold and found to be closely related to differences in electrical behavior of subepicardial and subendocardial tissue and myocytes with respect to frequency-dependent action potential changes, pharmacological sensitivity, and response to extracellular ion changes or ischemia.1 9 10 11

Although the exact roles of the transient outward current for action potential duration and processes of excitation-contraction coupling are complex12 and remain to be clarified, the transient outward current clearly exerts a significant effect on the height of the early plateau, thus influencing activation of other plateau currents that control repolarization. Furthermore, there is increasing evidence that the transient outward current may play an important role as mediator of neurohumoral influences on electrical properties of the myocytes.13 14 15

We previously reported the presence of a large calcium-independent transient outward current in myocardial cells isolated from central parts of the left ventricular wall of the human heart.16 17 18 Action potential recordings from other layers of the left ventricular wall indicated marked differences in the size of the notch of the early plateau (phase 1), strongly suggesting regional heterogeneity of the transient outward current in the human heart.1 17 19 Differences in current density of Ito1 in human myocardium were described recently for myocytes isolated from the epicardial and endocardial third of the left ventricular wall, but only a small difference in Ito1 density (1.54-fold) was found for nonfailing myocardium.20 In the present study, tissue sampling has been restricted to the subendocardial and subepicardial layers (up to 1-mm depth) of the left ventricular anterior wall. The data indicate not only an approximately fourfold larger Ito1 in subepicardial cells but also a much slower time course of recovery from inactivation in subendocardial cells, a feature distinguishing them from the very fast recovery in subepicardial cells. In addition, kinetic data at 35°C are presented to allow assessment of the role of Ito1 for electrical heterogeneity of the left ventricular wall.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
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Patients
Cells were prepared from 6 undiseased donor hearts (mean age, 41.6 years; range, 16 to 51 years) that were not transplanted for technical reasons and from 11 hearts of patients with end-stage heart failure caused by dilated cardiomyopathy (mean, 41.4 years; range, 17 to 62 years; 3 women; cardiac index, 2.21±0.19 L·min-1·m-2; ejection fraction, 23.6±2.2%). All patients with dilated cardiomyopathy received digoxin and diuretics and were under vasodilator therapy. No catecholamines, ß-adrenoceptor blocking drugs, calcium antagonists, or antiarrhythmic agents were given 48 hours before the operation. Informed consent was obtained before organ explantation. The protocol was approved by the Ethical Review Board of the University of Munich.

Cell Isolation
The isolation procedure was described in detail previously.21 A segment of the myocardium approximately midway between the apex and base of the left ventricular anterior wall was excised, together with an arterial branch of the left anterior descending coronary artery. This wall segment was then perfused through this arterial branch: 30 minutes with a nominally Ca2+-free modified Tyrode's solution ([mmol/L] NaCl 138, KCl 4, MgCl2 1, glucose 10, NaH2PO4 0.33, and HEPES 10; pH 7.3 was adjusted with NaOH at 37°C), followed by 40 minutes with the same solution with added collagenase (type II, 70 mg/50 mL, Worthington Biochemical Corp) and protease (type XIV, 6 mg/50 mL, Sigma Chemical Co). Finally, the enzyme was washed out for 15 minutes with modified Tyrode's solution that contained 200 µmol/L Ca2+. After this enzymatic digestion, the tissue still had its anatomic structure, so that identified regions of the myocardial wall could be sampled. Subepicardial and subendocardial myocytes were obtained by removing a thin layer (maximal thickness, {approx}1 mm) from the subepicardial and subendocardial surface with a scalpel. On the basis of a thickness of the left ventricular wall in this area of 8 to 11 mm, the layers used correspond to {approx}1/10th of the left ventricular wall. The distance of the two layers can be estimated to be between 7 and 10 mm. The tissue slices were cleared of myocytes of other regions sticking to the outer surfaces by washing with Tyrode's solution and then processed separately. After the tissue was cut into small pieces with scissors, the myocytes were disaggregated by mechanical agitation and, after filtration through a nylon mesh, stored at room temperature in Tyrode's solution containing 2.0 mmol/L Ca2+.

Living cell yield was {approx}5% to 8%. Only cells with clear cross striations without spontaneous contraction or significant granulation were selected for experiments. When field stimulated, these myocytes contracted as confirmed visually.

Solutions
For action potential recordings, cells were superfused at 35°C with a modified Tyrode's solution containing (mmol/L) NaCl 138, KCl 4, CaCl2 2.0, NaH2PO4 0.33, MgCl2 1, glucose 10, and HEPES 10; pH was adjusted to 7.3 with NaOH. For voltage-clamp experiments, 10 µmol/L tetrodotoxin was routinely added to reduce the fast sodium current (INa), facilitate voltage control, and minimize interference of current measurements by INa.22 In addition, a 100-ms prepulse to -60 mV from a holding potential of -80 mV was used to partially inactivate INa. To further minimize interference of INa with current readings, quantitative measurements of Ito1 were made close to the reversal potential of INa at 40 mV. The slow inward calcium current (ICa), which would interfere with measurements of Ito1 and might activate calcium-dependent currents,23 24 25 was blocked by addition of CdCl2 (0.3 mmol/L) to the extracellular solution, in line with our earlier studies and work by others.16 17 26 27 Although CdCl2 has been shown to cause a shift of inactivation and activation parameters of the transient outward current in the depolarizing direction,28 the alternative use of high concentrations of organic calcium channel blockers would similarly have been complicated by their effects on the density and kinetics of Ito1.29 30 Experimental conditions in voltage-clamp experiments were chosen to minimize calcium-activated outward currents by inclusion of EGTA to buffer [Ca2+]i and omission of Na+ from the intracellular solution to inhibit calcium influx through Na-Ca exchange and block of ICa by external Cd2+. Voltage-clamp experiments were performed at room temperature (21°C to 23°C). Under these recording conditions, no evidence for a contribution of chloride currents to the transient or pedestral outward current was found in previous studies.16 When recordings of kinetic parameters of Ito1 were made at 35°C, they are explicitly identified in the "Results" section. Fresh 4-aminopyridine was dissolved right before use in Tyrode's solution.

For current recordings, cells were dialyzed internally for at least 2 minutes with the patch electrode with (mmol/L) potassium aspartate 120, KCl 10, MgCl2 2, HEPES 10, EGTA 5, and MgATP 2; pH was adjusted to 7.2 with KOH. After this time, contraction on depolarization had ceased even in the absence of external Cd2+. For action potential recordings, an identical internal solution was used, supplemented by 5 mmol/L NaCl.

Recording Techniques
Experiments were carried out by standard single microelectrode whole-cell patch-clamp recording techniques31 with an Axopatch 200 A amplifier (Axon Instruments). Microelectrodes were pulled from borosilicate glass and had resistances of 2.0 to 3.0 M{Omega}. Series resistance was compensated as much as possible (30% to 80%). Analog filtering of current recordings was at 3 kHz. Currents were digitized at 2 to 10 kHz (unless stated otherwise) and stored for off-line analysis. Action potentials were recorded in current clamp mode. Cell capacitance was calculated by applying 5-mV steps from a holding potential of -80 mV in the hyperpolarizing direction and integrating the current required to charge the membrane when stepping back to -80 mV. No significant time-dependent currents were observed at this voltage. The temperature of the superfusing solution was measured with an NiCr-Ni microsensor.

Data Analysis
Fits to Boltzmann distribution and exponential kinetics were obtained by nonlinear least-squares techniques; goodness of fit for single exponential current decay was judged by visual inspection and {chi}2 testing as previously described.16 With the results (mean±SEM), the number of cells used is given, followed by a slash and the number of hearts from which the cells were derived. The Mann-Whitney nonparametric test was used for statistical evaluation, and values of P<.05 were considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
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Action potentials recorded in current clamp mode from subepicardial and subendocardial myocytes revealed a prominent notch in all cells of subepicardial origin. Action potential duration tended to be shorter in subepicardial cells (subepicardial, 424±20.9 ms, n=7/5; subendocardial, 486.2±32.6 ms, n=7/3; P=NS); resting membrane potential was similar in both groups (subepicardial, -79.5±0.48 mV; subendocardial, -78.8±1.38 mV; P=NS). The plateau of the action potential was consistently lower in subepicardial than in subendocardial cells (subepicardial: maximum plateau voltage after the notch at phase 1, 23.7±3.1 mV, n=7/5; subendocardial: voltage during plateau phase at dV2/dt2=0, ie, the transition from early repolarization to final repolarization, 36.0±3.06 mV, n=7/3; P<.05; see Fig 1Down, as well as Figs 8Down and 9Down). The notch in subepicardial cells, measured as the difference between the minimum of the notch and the plateau voltage, was 8.61±1.6 mV (n=7/5).



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  		Figure 1. Plots showing the differences in the action potential shape of human ventricular myocytes of subepicardial (A) and subendocardial (B) origin. Subepicardial myocytes displayed a clear notch at phase 1 of the action potential, suggested to be related to the presence of a prominent transient outward current. The notch was absent in subendocardial myocytes. Action potential duration tended to be shorter in subepicardial myocytes. Furthermore, the plateau of the action potential was consistently lower in subepicardial than in subendocardial myocytes. Action potential duration was as follows: subepicardial, 424±20.9 ms, n=7 cells from 5 hearts; subendocardial, 486.2±32.6 ms, n=7/3; P=NS. Resting membrane potential was -79.5±0.48 and -78.8±1.38 mV for subepicardial and subendocardial, respectively (P=NS). Action potential plateau for subepicardial was 23.7±3.1 mV; for subendocardial, 36.0±3.06 mV; P<.05. Stimulation frequency, 1 per 30 seconds; Vm, membrane potential; temperature, 35°C.



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  		Figure 8. Plots showing rate-dependent changes of the action potential in subepicardial and subendocardial myocytes. After 30 seconds at the resting membrane potential of the cell, action potentials were elicited in current clamp mode at a frequency of 2 Hz. Every other of the first 11 action potentials is shown for a subepicardial (A) and subendocardial (B) cell. Beat-dependent shortening of the action potential was accompanied by a slight increase in plateau voltage in subepicardial and subendocardial myocytes. A clear change in the depth of the notch at phase 1 of the action potential was not observed in subepicardial cells, consistent with the very fast recovery of Ito1 at physiological temperatures (inset; 35°C, n=12 cells from 3 hearts). The first action potential in the train is denoted by 1, the 11th by 11. The upstroke of membrane voltage on the right of each panel is the beginning of the next action potential.



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  		Figure 9. Plots showing the restitution of the action potential in subepicardial and subendocardial cells. To determine the recovery of action potential shape and duration during premature depolarizations in subepicardial and subendocardial myocytes, action potentials were elicited at increasing intervals beginning shortly before final repolarization of the preceding action potential. Restitution of the action potential shape is shown for a subepicardial (A) and subendocardial (B) myocyte. Action potential duration of premature depolarizations was abbreviated initially but quickly approached the duration of the preceding action potential in both subepicardial and subendocardial cells. Restitution of the notch at phase 1 of the action potential proceeded very rapidly in subepicardial cells, with no significant changes in the depth of the notch at phase 1 after more than 20 ms of repolarization (inset). The notch at phase 1 of the action potential was absent in subendocardial myocytes. The variable height of the phase 0 overshoot in A is due to a digitization frequency (1 kHz) insufficient to adequately resolve phase 0 of the action potential and does not reflect any physiological event.

For assessment of the density of Ito1 in subepicardial and subendocardial myocytes, current-voltage relations for the transient outward current were obtained by applying depolarizing pulses to voltages from -30 to 80 mV. To allow adequate recovery from inactivation, the pulse frequency was set to 1 per 20 seconds. In five subendocardial cells, no significant increase in Ito1 was observed when the holding time at -80 mV was prolonged from 20 to 90 seconds (at 20 seconds, the value was 96.3% of the value at 90 seconds), indicating that recovery was essentially complete after 20 seconds. Ito1 was measured as the difference in current between the peak of the transient outward current and the maintained current component at the end of the depolarizing clamp pulse. Current densities in myocytes from nonfailing hearts, calculated by dividing the transient outward current by the capacitance of the myocyte, are given as current-voltage relations of Ito1 in subepicardial and subendocardial cells in Fig 2Down. Densities of Ito1 were strikingly different in subepicardial and subendocardial cells, being about four times larger in cells of subepicardial origin (difference between peak current and maintained current at the end of the pulse to 40 mV at room temperature: subepicardial, 10.6±1.08 pA/pF, n=12/5; subendocardial, 2.63±0.31 pA/pF, n=22/5; P<.0001; nonfailing hearts; Table 1Down). Membrane capacitance of the myocytes was not different in the two groups (subepicardial, 184.5±20.5 pF; subendocardial, 174.2±16.9 pF; P=NS). In the same cells, the maintained, late component of the outward current was similar in subepicardial and subendocardial cells (at 40 mV and room temperature: subepicardial, 2.14±0.58 pA/pF; subendocardial, 1.49±0.17 pA/pF; P=NS). In myocytes derived from failing hearts, the density of Ito1 (difference between the peak and maintained currents at the end of the pulse to 40 mV) was significantly smaller in subepicardial cells (7.81±0.53 pA/pF, n=20/5; P=.012) but not different in subendocardial myocytes (2.01±0.23 pA/pF, n=14/4; P=.25; see Table 1Down).



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  		Figure 2. Plots showing the voltage dependence of the current density of Ito1. The panels denote (from top to bottom) voltage protocol, original current recordings, and current densities in subepicardial myocytes of nonfailing hearts (A, {bullet}, n=12 cells from 5 hearts, mean±SEM) and subendocardial myocytes (B, {circ}, n=22/5). Holding potential was -80 mV, and a 100-ms prepulse to -60 mV was applied before the membrane voltage was stepped from -30 to 80 mV for 500 ms. Ito1 was measured as the difference between absolute peak current of Ito1 and maintained current at the end of the clamp pulse. Pulse rate, 1 per 20 seconds; room temperature.


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  		Table 1. Current Density of Ito1 in Myocytes From Nonfailing and Failing Hearts

Steady state inactivation parameters of Ito1 were obtained with a double-pulse protocol with 1-second prepulses to voltages from -60 to 40 mV from a holding potential of -80 mV as previously described.16 Peak currents at the test pulse to 60 mV were normalized to the maximal transient outward current elicited by the test pulse and fitted individually for each cell by a single Boltzmann distribution, resulting in a slightly more negative V1/2 in subendocardial cells (V1/2, -17.6±1.02 mV; slope factor, 8.9±0.92 mV, n=10/4) compared with subepicardial cells (V1/2, -9.5±0.38 mV, P=.0012; slope factor, 5.1±0.44 mV, P=.0054; n=8/3). Activation of the transient outward current was observed first around 0 mV in both subepicardial and subendocardial cells with a similar current-voltage relation in cells of both origins. Activation parameters of Ito1 (calculated by assuming a linear open channel conductance and a reversal potential of -58 mV16 ) were not different in subepicardial (V1/2, 29.1±1.2 mV; slope factor, -12.9±1.1 mV; n=12/5) and subendocardial myocytes (V1/2, 32.0±1.11 mV, P=NS; slope factor, -14.9±0.81 mV, P=NS; n=22/5). In myocytes from failing hearts, activation parameters in subepicardial (V1/2, 25.7±1.5 mV; slope factor, -13.4±0.74 mV; n=14/4) and subendocardial cells (V1/2, 31.6±1.61 mV; slope factor, -15.0±1.1 mV; n=11/4) were not different from the corresponding values in nonfailing myocardium (Fig 3Down). It should be noted that the V1/2 -values are expected to shift to more positive potentials because of the use of the divalent ion Cd2+ to block ICa.28 Determination of potassium selectivity from tail currents was not successful owing to the small size of decaying tails in endocardial cells that could not be measured reliably. In both subepicardial and subendocardial cells, Ito1 was sensitive to 4-aminopyridine with almost complete block of the transient outward current at 3 mmol/L 4-aminopyridine (depolarization to 40 mV for 300 ms at 0.1 Hz). The nature of the small maintained outward current component remains to be clarified. The insensitivity of this current component to 4-aminopyridine in human ventricular myocytes16 suggests that it is not due to the ultrarapid delayed rectifier current described recently.32 However, the complex state-dependent blocking properties of 4-aminopyridine with respect to Ito133 may obscure a contribution of this current to the observed late maintained outward current.16 An IK-delayed rectifier current would not be expected to contribute significant outward currents at room temperature.



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  		Figure 3. Plots showing the voltage dependence of activation of Ito1. A, Nonfailing myocardium; B, failing myocardium. Voltage-dependent activation was calculated from IV relations assuming a linear open channel conductance. (Subepicardial myocytes, {bullet}; nonfailing, n=12 cells from 5 hearts; failing, n=14/4; subendocardial myocytes, {circ}; nonfailing, n=22/5; failing, n=11/4.) Solid lines represent the activation curves calculated from mean values for half–activation voltage and slope obtained by fitting a Boltzmann distribution to the data in each individual cell of the respective cell population.

The time course of inactivation of the transient outward current, which has been shown to be largely independent of voltage in human myocytes and myocytes of several other species,16 24 28 was not different in subepicardial and subendocardial myocytes (Fig 4Down and Table 2Down). To allow estimation of the time-dependent influence of Ito1 on action potential configuration at physiological temperatures, inactivation kinetics were studied at 35°C. Determination at physiological temperatures is especially important because the kinetic properties of the transient outward current are known to be highly temperature dependent.34 The results indicate that inactivation is speeded up by a factor of six at 35°C compared with room temperature (data summarized in the legend to Fig 4Down).



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  		Figure 4. Plots showing the inactivation time course of Ito1 in subepicardial and subendocardial myocytes at room temperature and 35°C. Original current traces (top) and semilogarithmic plots of Ito1 in subepicardial (A and B) and subendocardial (C and D) myocytes. Current at the end of a 500-ms depolarization to 40 mV was subtracted before normalization of current. Traces were aligned along the time axis to superimpose at the time of peak Ito1. Current decay followed a monoexponential time course with similar rate constants in both subepicardial and subendocardial cells but was about six times faster at 35°C than at room temperature (RT). Traces are from different cells. I/Imax refers to the transient current component only (difference between peak current and maintained current at the end of the pulse). The apparently larger maintained current in C is due to the relatively small transient component in subendocardial cells. Mean values for the inactivation time constant were as follows: at room temperature: subepicardial, 48.7±3.0 ms, n=17 cells from 6 hearts; subendocardial, 44.76±1.96 ms, n=18/6, P=NS; at 35°C: subepicardial, 7.86±0.25 ms, n=7/3; subendocardial, 7.02±0.42 ms, n=7/3, P=NS.


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  		Table 2. Time Course of Recovery From Inactivation (-80 mV)

For analysis of frequency-dependent properties of the transient outward current, depolarizing steps to 40 mV were given for 250 ms from a holding potential of -80 mV at frequencies up to 2 Hz. When comparing the fifth pulse in a train of pulses at 2 Hz to the first pulse given after a 30-second rest at -80 mV, only a small reduction of peak transient outward current was observed in subepicardial cells (to 92.3±1.9%, n=16/4), whereas Ito1 was almost completely suppressed in subendocardial cells (reduction to 13.2±2.1%, n=15/4, P<.0001; Fig 5Down). Rate-dependent reduction of Ito1 was not different in myocytes isolated from failing myocardium (subepicardial, 90.8±1.9%, n=6/3, P=.36; subendocardial, 10.1±2.3%, n=6/3, P=.39).



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  		Figure 5. Plots showing the different rate-dependent reduction of Ito1 in subepicardial and subendocardial cells. After a 30-second rest at -80 mV, depolarizing clamp pulses were given to 40 mV at a 2-Hz frequency for 250 ms. The first five consecutive current traces are shown for cells of subepicardial (A) and subendocardial (B) origin. Although in subepicardial cells the transient outward current displayed only a minimal frequency-dependent reduction, {approx}90% of Ito1 was suppressed in subendocardial cells in subsequent depolarizations.

This different rate-dependent reduction of Ito1 suggested differences in recovery from inactivation in cells of subepicardial and subendocardial origin, prompting the study of restitution of Ito1. At room temperature (21°C to 23°C), recovery of the transient outward current from inactivation was very fast in subepicardial cells (time constant of main component, 46.1 ms), similar to what has been observed in myocytes derived from central parts of the left ventricular myocardium.16 In contrast, recovery from inactivation in subendocardial cells was slow, with only half of the current available after {approx}1800 ms (Fig 6Down). To obtain time parameters of recovery kinetics, nonlinear fitting was performed with biexponential functions, based on earlier observations in myocytes of human myocardium and several other species that two exponentials usually were required to adequately fit the recovery kinetics of Ito1.12 14 16 35 36 The resulting values, summarized in Table 2Up, indicated that >90% of the recovery of Ito1 in subepicardial cells follows a fast time course, whereas about 90% of the recovery follows a slow time course in subendocardial myocytes. To allow assessment of availability of Ito1 during the cardiac cycle at physiological temperatures, recovery parameters were determined at 35°C. The differences in speed of recovery of subepicardial and subendocardial myocytes were similar at 35°C (Fig 7Down). However, the recovery processes of the major components in subepicardial cells (fast component) and subendocardial cells (slow component) were faster by factors of >5 and 3.7, respectively, at 35°C compared with at room temperature (Table 2Up). A comparison of the time constants of the small components did not appear to be useful because of the large errors expected for the values obtained from fitting.



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  		Figure 6. Plots showing time course of recovery from inactivation in subepicardial and subendocardial cells at room temperature. Recovery from inactivation was determined with a standard double-pulse protocol with depolarizations to 40 mV; the holding voltage between pulses was -80 mV, and depolarizations were to 40 mV for 400 ms. A, Original current recordings in a subepicardial myocyte, showing very fast recovery of Ito1 in these cells. B, Current recording in a subendocardial myocyte. C and D, Recovery from inactivation in subepicardial ({bullet}, mean±SEM, n=9 cells from 3 hearts) and subendocardial ({circ}, n=11/4) myocytes at room temperature. The initial 500 ms of recovery is replotted in D on an expanded time scale. Capacitive transients were partially removed for clarity. Data are from nonfailing hearts only.



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  		Figure 7. Plots showing time course of recovery from inactivation in subepicardial and subendocardial cells at 35°C. This figure corresponds to Fig 6Up at 35°C, displaying original current traces of subepicardial (A) and subendocardial (B) myocytes. Recovery from inactivation was extremely rapid in subepicardial cells, about fivefold faster than at room temperature. Similarly, recovery from inactivation in subendocardial myocytes at 35°C was faster by a factor of about 3.7 compared with room temperature. Note that the time scales in A and B are different from those in Fig 6AUp and 6BUp. C and D, Recovery from inactivation in subepicardial ({bullet}, mean±SEM, n=9 cells from 3 hearts) and subendocardial ({circ}, n=10/3) myocytes at 35°C. D, Replot of the first 500 ms of C on an expanded time scale. To facilitate comparison, the time scales in C and D are identical to those in Fig 6Up. Capacitive transients were partially removed for clarity.

Frequency-dependent changes of the action potential in subepicardial and subendocardial myocytes, suggested to be influenced by differences in Ito1,12 were evaluated in current clamp mode at 35°C. Stimulation (at 150% of threshold) and recording of intracellular potentials was through the patch electrode. Increasing the stimulation frequency from 1 per 30 seconds to 2 per 1 second caused the action potential to shorten in both subepicardial and subendocardial myocytes, with a slight increase of the early plateau voltage (Fig 8Up). The depth of the notch at phase 1 did not change significantly in the train of pulses (n=12/3; Fig 8Up). In endocardial cells, a notch at phase 1 of the action potential could not be detected, even when the interval between depolarizations was made long enough to allow essentially complete recovery of Ito1 from inactivation (30 seconds).

In the dog, restitution of phase 1 of the action potential (which is considered to be related to Ito1 density3 ) was found to be biphasic, with reduced availability of Ito1 during early recovery of the action potential plateau.1 3 9 In dog heart subepicardium, some important pathophysiological phenomena, such as supernormal conduction1 or phase 2 reentry,9 may be linked in part to delayed reactivation of Ito1. In contrast, the fast reactivation time course of Ito1 in human subepicardial cells suggests rapid restitution of the phase 1 action potential notch and plateau characteristics. Therefore, restitution of action potential characteristics was studied in human subepicardial and subendocardial myocytes. The data (Fig 9Up) indicate that the notch at phase 1 of the action potential reappeared rapidly in subepicardial cells, with no significant changes in the depth of the notch at phase 1 in action potentials elicited after >20 ms of repolarization. This is consistent with the rapid time course of recovery of Ito1 in subepicardial cells (time constant {tau}=7.86 ms at 35°C; Fig 7Up). In subendocardial cells, where the notch at phase 1 was absent, restitution of the final shape of the action potential proceeded with an equally fast time course. It should be noted that experimental conditions were selected to preferentially study Ito1, so other ionic currents possibly important for physiological modulation of the action potential height, shape, and duration may have been reduced or eliminated, especially Ca2+-activated currents.


*    Discussion
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*Discussion
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This report characterizes regional densities and properties of Ito1 in human ventricular myocytes. The results indicate large differences in density of Ito1 in subepicardial and subendocardial layers of the left ventricular wall of the human heart. Detailed experimental evidence is provided to show that rate-dependent properties of Ito1 are distinctly different in subepicardial and subendocardial layers of human left ventricle. Action potential recordings were performed for assessment of the role of Ito1 for transmural voltage gradients and frequency dependence of the action potential configuration. However, limitations caused by the experimental conditions favoring dissection of Ito1 (use of dialyzing pipettes with alteration of the intracellular constituents and, most importantly, suppression of Ca2+ transients by EGTA) must be considered when the action potential data are extrapolated to physiological conditions.

Differences in Action Potential Characteristics
The characteristic difference in action potential shape between subepicardial and subendocardial cells was the presence of a notch at phase 1 of the action potential in myocytes of subepicardial origin (Figs 1Up, 8Up, and 9Up). A similar difference was noted previously in dog,4 cat,5 rat,7 and rabbit8 left ventricular myocytes in which this notch could also be related to the presence of a prominent transient outward current in cells of subepicardial origin. Furthermore, action potential duration tended to be shorter and the voltage level of the plateau to be lower in subepicardial cells, the latter reflecting the early repolarizing effect of the transient outward current. Because the rapid inactivation kinetics of the transient outward current at physiological temperatures ({tau} <10 ms at 40 mV; Fig 4Up) make a direct contribution of the transient outward current to the final phase of repolarization (ie, after {approx}300 ms) very unlikely, the transient outward current probably exerts its influence on action potential duration indirectly by setting the voltage of the early plateau phase.1 12

Current Density of Ito1
For nonfailing myocardium, current density of the Ito1 was larger by a factor of about four in myocytes of subepicardial compared with subendocardial origin. This difference and the absolute densities of Ito1 in subepicardial and subendocardial myocytes were in a similar range as the values reported for dog left ventricular wall at comparable voltages (subepicardial, 29.0±13.7 pA/pF; subendocardial, 5.59±3.19 pA/pF at 70 mV8 ). It should be noted that the myocytes used in this study were derived from a very similar area of the left ventricular wall. In feline myocytes, current densities three to four times larger (102±47.7 pA/pF at 80 mV) were reported in subepicardial myocytes, whereas the current density in subendocardial cells was similar (3.3±3.3 pA/pF).7 In contrast, the difference reported for rabbit left ventricle was markedly smaller and significant only at very positive potentials.4 In a recent study in human ventricular myocytes from segments spanning approximately one third of the epicardial and endocardial wall, current densities were smaller for cells of subepicardial but similar for subendocardial origin, possibly related to the differences in cell origin and experimental conditions, including the use of 2,3-butanedione monoxime for myocardial preservation.20

Frequency-Dependent Properties
Despite the electrophysiological similarities in subepicardial and subendocardial myocytes, Ito1 in subepicardial and subendocardial myocytes is clearly distinguished by the time course of recovery from inactivation. When overall recovery from inactivation of Ito1 is assessed, only about 35 ms (at room temperature) was required for half-maximal recovery of Ito1 compared with {approx}1800 ms in subendocardial cells. When a biexponential function is fit to the time course of recovery from inactivation, >90% of the recovery from inactivation followed a fast time course in epicardial cells but a very slow time course in subendocardial cells (Figs 6Up and 7Up and Table 2Up). Biphasic recovery of whole-cell transient outward current from inactivation was observed previously in myocytes derived from central parts of the left ventricular wall16 and atrial myocytes36 of the human heart and several animal species.12 14 35 In addition, a wide range of recovery rates has been reported for the transient outward current in ventricular myocytes of different animal species, with fast recovery in rat37 but relatively slow kinetics in dog or rabbit.35 36 38 This indicates a large variability of this parameter in different species and illustrates the difficulties in extrapolating data from animal to human myocardial tissue.

Because the current densities given in Fig 2Up were measured at a low pulse rate selected to allow essentially complete recovery of Ito1 from inactivation (1 per 20 seconds), the transmural difference in current density of Ito1 will become even larger with increasing frequency owing to incomplete recovery of Ito1 in subendocardial cells. In subepicardial cells, on the other hand, Ito1 will contribute with nearly its maximal density to repolarization, largely independent of frequency at physiological heart rates. This very fast recovery of Ito1 in subepicardial myocytes resembles findings in human atrial myocytes, where Ito1 was also found to be essentially rate independent at physiological heart rates.36

Although Ito1 is small in subendocardial myocytes, it may nevertheless contribute to repolarization of the early plateau phase because even small currents can have a significant effect on the balance of currents during the plateau of the action potential. With about half of Ito1 available within 500 ms at 35°C, a significant frequency-dependent variability of Ito1 is expected in the subendocardial layer within the physiological range of heart rates. Furthermore, although the current densities reported here are from the two extreme surfaces of the left ventricular myocardium, the change in current densities from subepicardial to subendocardial is most likely a continuous process, with increasing density of the fast recovering transient outward current and decreasing density of the slowly recovering subtype from subendocardial to subepicardial layers, involving regions with transitional electrophysiological characteristics, as suggested from action potential recordings in dog left ventricle.39 This is also supported by previous data from central parts of the left ventricular wall,16 in which Ito1 current densities and the fraction of current recovering at a fast rate had values between those of subepicardial and subendocardial cells found in this study.

A recent study on Ito120 in human myocytes also presented support for our previous report of a slow recovery process in subendocardial myocytes,40 even though the results were from limited data in a few cells derived from the endocardial one third of the myocardial wall. However, recovery was still found to be relatively fast, reaching {approx}50% of its final value within 100 ms (compared with {approx}10% in this study; Fig 6Up). In addition, some analyzed cells apparently displayed a biphasic decay of the outward current with a second slow phase of current decay (Fig 9Up in Reference 20). This is atypical for Ito116 34 and suggests the presence of additional unidentified current components that may have contributed to apparently nonrecovering current components as proposed.20

Influence of Underlying Pathology
Several studies have indicated that the density of the transient outward current may be subjected to alterations in cardiac hypertrophy41 or failure,17 during ischemia,42 after myocardial infarction,43 or under the influence of an altered thyroid state.44 In a previous study in myocytes derived from central parts of the left ventricular wall, Ito1 was significantly reduced (by 37%) in myocytes derived from failing compared with nonfailing myocardium.17 A similar finding has now been made for myocytes in the subepicardial layer: Ito1 density was 26.4% smaller for cells from failing myocardium. Current density of Ito1 in myocytes from the subendocardial layer was not different for failing and nonfailing hearts. These findings differ from those of Wettwer et al,20 who reported a decrease in current density of Ito1 in subendocardial but not subepicardial cells in heart failure. However, tissue sampling was not as strictly defined in their study in that no specific ventricular site was selected and a thick wall segment was included to obtain "subepicardial" and "subendocardial" myocytes (up to 1/3 of the thickness of the wall on each side). This allows a large variability of the origin of the myocytes (data in dog left ventricle indicate that important density changes of Ito1 may occur in as little as 1/10th of the thickness of the myocardial wall45 ), which might have significantly influenced the results, especially because the data for subepicardial cells of nonfailing myocardium were obtained from only two hearts. It also may have contributed to the small current density in nonfailing subepicardial cells (6.8±0.4 pA/pF at 60 mV) compared with our data (15.6±1.7 pA/pF at 60 mV) and their previous value in nonselected left ventricular cells of nonfailing myocardium (8.7±1.0 pA/pF at 60 mV37 ). This, together with the very small difference of Ito1 in subepicardial to subendocardial cells in nonfailing myocardium (1.54-fold; this study, 4-fold; dog left ventricle, 5.2-fold at 70 mV8 ) and the relatively fast recovery in their subendocardial cell preparation ({approx}50% within 100 ms), suggests that the inner and outer 1/3 of the human left ventricular wall may not closely reflect the properties of thin subepicardial and subendocardial layers. Precautions against regional influences of tissue sampling are especially warranted for the subendocardial layer because the small size of Ito1 and the rapid increase of Ito1 toward the deeper subendocardium render data on current densities from this layer very sensitive to the contaminating influence of cells from deeper layers.

Implications for Electrophysiology of Human Heart
The large gradient in current density of the transient outward current in myocytes from subendocardial to subepicardial layers suggests the presence of a pronounced electrical heterogeneity in the left ventricular wall of the human heart. The relevance of the measurements in isolated myocytes for myocardial tissue is strengthened by the similarity of the findings in isolated canine tissue and myocytes obtained from identical myocardial regions8 and by action potential recordings in human subepicardial tissue that also indicated the presence of a transient outward current.46

The importance of the transient outward current for heterogeneity of electrical behavior and pharmacological responses of myocardium was demonstrated recently in dog heart.1 In tissue and myocytes of canine left ventricular wall, differences in action potential characteristics, rate dependence of action potential restitution, and sensitivity to extracellular ion changes (potassium and Ca2+) were shown to be closely related to the different densities of Ito1.9 Furthermore, the high density of the transient outward current was found to contribute significantly to the increased sensitivity of epicardial myocardium to electrical depression during ischemia.11 Because the transient outward current appears to be present in similar densities and gradients in subepicardial and subendocardial layers of the human left ventricular wall as in the dog, some findings may be applicable to the human heart under similar conditions. However, the rapid recovery of Ito1 in human subepicardial cells should be remembered when phenomena possibly related to reduced early availability of Ito1 in dog, such as supernormal conduction1 or phase 2 reentry, are considered.9

In addition, drugs blocking Ito1 will differentially influence electrophysiological properties in subepicardial and subendocardial myocytes. This has been studied for 4-aminopyridine, considered to be a relatively selective blocker of the transient outward current, which exerts markedly different effects in subepicardial compared with subendocardial cells in canine left ventricle on action potential duration, rate-dependent effects, ischemia-induced electrical depression, and other electrical phenomena considered to be related to Ito1.1 Different densities of Ito1 also may cause differential sensitivity to reduction of inward currents conducting at a similar time during the action potential, such as block of the sodium channel. For flecainide, this has been implicated to promote electrical heterogeneity and dispersion of repolarization as possible mechanisms of proarrhythmic effects.1 10 47 Because several antiarrhythmic drugs, including quinidine,48 propafenone, flecainide,49 50 tedisamil,51 and the antidepressant imipramine,52 have recently been shown to block Ito1 in possibly therapeutically relevant concentrations, the density gradient of Ito1 may have immediate clinical implications in that these compounds might differentially affect electrical properties of subepicardial and subendocardial myocardium.

In conclusion, a large gradient in density- and rate-dependent properties of the transient outward current exists in different layers of the left ventricular wall of the human heart causing a pronounced electrophysiological heterogeneity. Ito1 appears to be reduced in heart failure in subepicardial myocytes, similar to what has been found in central layers of human left ventricle. Even though other currents might also exhibit regional variability within the left ventricular wall, the large difference in density and properties suggests that the transient outward current is of primary importance for the regional heterogeneity of electrophysiological properties of the myocytes and the electrical behavior of the myocardium in human left ventricular wall.


*    Acknowledgments
 
This research was supported by a grant of the Deutsche Forschungsgemeinschaft.

Received December 7, 1994; revision received August 1, 1995; accepted August 14, 1995.


*    References
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*References
 

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