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(Circulation. 1997;96:1266-1274.)
© 1997 American Heart Association, Inc.

Articles

Regional Alteration of the Transient Outward Current in Human Left Ventricular Septum During Compensated Hypertrophy

Patrick Bailly, MD; Jean-Pierre Bénitah, PhD; Maud Mouchonière, PhD; Guy Vassort, DSc; ; Paco Lorente, MD

From the Département de Chirurgie Cardiovasculaire, Hôpital Gabriel Montpied, Clermont-Ferrand (P.B.), and U 390 INSERM, CHU Arnaud de Villeneuve, Montpellier (G.V., P.L.), France. J.-P.B. is currently a postdoctoral fellow in the Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Md. M.M. is currently a postdoctoral fellow in the Institut National de Recherche Agronomique, Theix, France.

Correspondence to Dr Paco Lorente, U 390 INSERM, CHU Arnaud de Villeneuve, 34295 Montpellier, France. E-mail paco{at}u390.montp.inserm.fr

	Abstract

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Background A large calcium-insensitive transient outward current (I_to) has been recorded in atria, left ventricular (LV) free wall, and right ventricular septal subendocardium of the human heart. Recent studies suggested a major contribution of this current to the electrical heterogeneity of the heart. However, no data have been reported on the distribution of I_to density within the LV septal wall from compensated human LV hypertrophy.

Methods and Results Microelectrode and patch-clamp techniques were used to record action potentials and I_to in myocytes isolated from superficial (<3 mm deep) and deep (3 to 6 mm deep) layers of LV septum from patients with aortic stenosis and compensated LV hypertrophy. Subendocardial specimens were also obtained from undiseased donor hearts. In none of the superficial subendocardial cells from diseased hearts was a macroscopic I_to recorded (n=42), whereas in cells from the same location from donor hearts, a typical I_to was clearly present, with a peak density of 5.88±0.78 pA/pF at +60 mV (n=4). However, in deep layers from patients with compensated LV hypertrophy, macroscopic I_to was present, with a peak density of 10.50±2.58 pA/pF at +60 mV (n=4). The absence of I_to in superficial septal cells from hypertrophied hearts was not due to a divalent cation–related shift of the current kinetics. Instead, extracellular Ca²⁺ removal induced an I_to-like current, possibly carried by K⁺ ions, with a peak density of 30.7±2.6 pA/pF at +60 mV (n=29). However, its magnitude, kinetics, and pharmacological characteristics did not allow identification of this current as the usual I_to.

Conclusions Both topography and pathology can be major modulating factors of the regional distribution of I_to density in human LV septum. Therefore, they may play a prominent role in determining electrical gradients within this region from which the early depolarization vectors start and the left-to-right activation sequence of the interventricular septum proceeds.

Key Words: action potentials • electrophysiology • hypertrophy • ventricles

	Introduction

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The Ca²⁺-insensitive 4-AP–sensitive I_to plays a major role in modulating cardiac electrical activity.^{1 2} It underlies phase 1 repolarization, and thus, by setting the voltage of the early plateau phase, it influences activation and inactivation of other plateau currents that control repolarization. It is also clear from several studies that I_to channels are potentially important targets for both neuromodulatory control³ and antiarrhythmic drug action.^{4 5 6} This current has been suggested to contribute significantly to the regional electrophysiological heterogeneity within the ventricular wall,^{7 8 9 10 11 12 13} a fact considered to be responsible for T-wave polarity.^{14 15} The heterogeneous distribution of I_to thus appears to be essential in causing the transmural electrical gradients necessary for proper repolarization of the cardiac action potential.^{1 16 17} It is expected that changes in I_to distribution and availability could be expressed in the ECG by typical J-wave and T-wave alterations and may lead to cardiac arrhythmias during evolving heart disease.¹ A better knowledge of the differential distribution of I_to among regions of the human heart therefore has considerable clinical relevance. Regional differences in I_to characteristics within human heart have recently been documented,^{16 18 19} but the issue of the regional distribution of I_to within the human ventricular septum has not yet been addressed. This region can be considered crucial in terms of cardiac depolarization, because the early ventricular excitation originates in the left septal surface and the initial movement of the depolarizing wave is mainly from left to right in human ventricular septum.²⁰

Modulation of electrical activity and I_to heterogeneity by cardiac hypertrophy has been investigated in several animal models^{12 21 22 23 24 25 26} ; from this, the consensus has emerged that in most cases pressure overload induces a decrease in I_to current density. With respect to human ventricular myocytes, it has been tentatively speculated that the inner layers of LV free wall are more prone to the depressing effect of heart failure on I_to than the outer tissue,¹⁹ but a significant decrease of I_to density has been reported only in subepicardial cells from failing compared with nonfailing human ventricles.¹⁶ We previously failed to find any evidence of I_to in human ventricular myocytes from patients with LV hypertrophy,²⁷ but no study has been published thus far on the regional distribution of this current in human compensated cardiac hypertrophy.

The aim of our study was to determine whether I_to density may be differentially distributed within human LV septum and to investigate the influence of pressure overload–induced cardiac hypertrophy in adult patients suffering from aortic stenosis without LV dysfunction. During our investigations, no evidence was found for the presence of macroscopic I_to in superficial subendocardial layers from hypertrophied LV septum, whereas I_to was clearly present in the same type of cells from nondiseased hearts. Furthermore, in those cells devoid of macroscopic I_to, removal of extracellular calcium was able to induce a large, rapidly activating and inactivating outward current that could not be attributed to some unmasked I_to.

	Methods

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Characterization of Patients
Ventricular tissue samples were obtained from 47 patients undergoing corrective cardiac surgery for acquired aortic stenosis with documented LV hypertrophy but without LV dysfunction. Patient age was 64.7±11.4 years (range, 50 to 84 years); 30 were women. All patients had clear evidence of LV hypertrophy: echocardiographic septal thickness was 14.3±1.1 mm (normal values, <10 mm). They were diagnosed as having compensated cardiopathy (NYHA class II; cardiac index, 2.41±0.46 L·min^-1·m^-2; ejection fraction, 0.57±0.23; LV end-diastolic pressure, 14.6±3.7 mm Hg; echocardiographic shortening fraction, 0.35±0.13) and hence were not receiving any cardiotonic drugs. All patients gave written informed consent to the study protocol, which was previously approved by the institutional committee on human investigation and complies with principles outlined in the Declaration of Helsinki. Results were compared with those obtained from two undiseased donor hearts that could not be transplanted for technical reasons (1 man, 51 years old; 1 woman, 60 years old; no therapy, died of cerebral trauma).

Tissue Sampling Technique
Endocardial LV septal myectomy was performed through the aortic orifice during circulatory arrest. Samples were taken from the muscular septum just beneath the commissure between left and right coronary leaflets of the aortic valve. Specimens (10 to 12 mm long, 5 to 7 mm wide, <3 mm thick, 0.1 to 0.6 cm³) consisted of scalpel shavings from the LV septal wall, in most cases superficial (<3 mm deep). In two cases showing large concentric hypertrophy, deeper samples were obtained from partial resection of functional muscle stenosis of the outflow tract: a 3-mm superficial layer was removed, and only the 3- to 6-mm-deep layer was used. After this procedure, the dimensions of deep samples were approximately the same as those of superficial samples. Specimens from the same location were obtained from donor hearts. Immediately after sampling, every piece of tissue was bathed in a cardioplegic solution bubbled with 100% O₂ at 19°C. Experimental procedures began within 15 minutes of removal. Half of the sample was used for conventional microelectrode and the other half for whole-cell voltage-clamp studies.

Studies on Syncytial Preparations
The preparations (1 to 2 mm long) were placed in a tissue bath and allowed to equilibrate for at least 2 hours while being superfused with an oxygenated (95% O₂/5% CO₂) Tyrode's solution (37±0.5°C, pH 7.35). The composition of the Tyrode's solution was (in mmol/L) NaCl 130, KCl 4, NaH₂PO₄ 1.2, NaHCO₃ 24, CaCl₂ 1.8, MgCl₂ 1, and glucose 5.6, with pH adjusted to 7.4 with NaOH. Tissues were stimulated at 500- to 5000-ms basic cycle lengths with rectangular stimuli (duration, 2 ms; intensity, 1.5 times diastolic threshold) delivered through Teflon-coated bipolar silver electrodes. Membrane potentials were recorded with 3 mol/L KCl–filled glass microelectrodes (15 to 20 M DC resistance) coupled through a Ag/AgCl electrode to a high-input impedance amplifier (Biologic VF102-IS100). Amplified signals were displayed on a digital storage oscilloscope (Nicolet 310) and stored for data analysis (NeuroCorder DR-484). After impalement, the stimulation rate was increased stepwise, and action potentials were recorded after complete stabilization of the signals. APDs were measured at 90% (APD₉₀) of repolarization.

Cell Isolation
Ventricular myocytes were isolated by an enzymatic dissociation method as previously described.²⁷ Briefly, chunks were incubated at 35°C for 30 minutes in a Ca²⁺-free Tyrode's solution supplemented with 300 IU/mL collagenase V, 4 IU/mL protease XXIV, and 1 mg/mL BSA (Sigma). Then the supernatant was removed and replaced by a fresh enzyme medium having the same composition but without protease. When the yield appeared to be maximal, minced tissue was strained through a 200-µm nylon mesh to remove debris and undigested tissue. Cells were then suspended in Ca²⁺-free Tyrode's solution and stored 1 hour at room temperature (20°C to 22°C) before the experiment was started. Only quiescent rod-shaped cells showing clear striations without significant granulation were used.

Single-Cell Recording Techniques
Macroscopic current recordings were obtained by standard whole-cell recording technique²⁸ with an Axopatch 1D amplifier (Axon Instruments) with a 100-M feedback resistance headstage. For stimulus protocol design and data acquisition, the 125-kHz Labmaster board and pClamp program V 5.5.1 (Axon Instruments) were used in connection with a personal computer. Microelectrodes pulled from soft glass capillary tubing (1.5- to 1.6-mm OD) had tip resistances ranging from 1 to 1.5 M. A Ag/AgCl pellet encased in a 3 mol/L KCl agar bridge was systematically placed in the bath and used as the ground reference electrode. Cell capacitance and series resistance were measured by application of +10- and -10-mV voltage steps from a -70-mV holding potential and calculated as previously described.²⁷ Series resistance was kept at <5 M (2.38±1.24; n=72) and was compensated by 60% to 80% without causing ringing. The electrode potential was adjusted to zero; this zeroing caused a positive voltage bias that was not corrected. Currents were low-pass filtered at 2 kHz, digitized at a sampling interval of 200 µs (unless otherwise stated), and stored for off-line analysis.

Solutions
The cardioplegic solution (transport solution) contained (in mmol/L) NaCl 147, KCl 20, MgCl₂ 16, glucose 6, and CaCl₂ 2 (pH 6.8 with KOH). For cell isolation and cell storage, the Ca²⁺-free Tyrode's solution had the following composition (in mmol/L): NaCl 120, KCl 4, MgCl₂ 1, HEPES 10, and glucose 6; pH was adjusted to 7.4 with NaOH. For whole-cell voltage-clamp experiments, the standard Tyrode's solution contained (in mmol/L) NaCl 130, KCl 4, CaCl₂ 2, MgCl₂ 1.1, mannitol 0.4, HEPES 25, and glucose 11; pH was adjusted to 7.4 with NaOH. To eliminate contamination by Na⁺ current²⁹ and exclude contribution of either Na⁺-activated potassium currents or transient currents generated by the Na⁺-K⁺ pump,³⁰ choline chloride or tetraethylammonium chloride was substituted in equimolar amounts for NaCl. The internal solution had the following composition (in mmol/L): KCl 120, MgCl₂ 1, Mg₂ATP 3, Tris-GTP 0.4, EGTA 10, HEPES 25, and glucose 10 (pH 7.2 with KOH). The slow inward current, which would overlap elicited I_to current and might activate calcium-dependent currents,^{31 32} was blocked by addition of 2 mmol/L CoCl₂ to the extracellular solution. Indeed, it has been demonstrated that CoCl₂ can cause significant positive shifts of steady-state activation and inactivation parameters of I_to,³³ but the alternative application of organic calcium channel blockers can be even more confusing because of their actions on the amplitude and kinetics of I_to.^{30 34} The addition of EGTA buffer to the internal solution was aimed at minimizing Ca²⁺-activated outward currents, and the lack of Na⁺ in the internal solution was also expected to inhibit Ca²⁺ influx through Na-Ca exchange. I_to could be inhibited by 3 mmol/L 4-AP. In some experiments, extracellular [Ca²⁺] was varied from 0 to 8 mmol/L, [K⁺]_o was increased to 8 mmol/L, and KCl was substituted by an equimolar amount of CsCl in both external and internal solutions.

Statistics
The results are expressed as mean±SEM. Fits to experimental data were performed by nonlinear least-squares techniques. The Mann-Whitney nonparametric test was used for statistical evaluation. Significance was assumed when P<.05.

	Results

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Action Potential
At 37°C, action potentials recorded from superficial subendocardium specimens isolated from undiseased septal myocardium were characterized by a small notch between phases 1 and 2. Phase 3 repolarization was steep, and APD₉₀ was 384±23 ms (n=4, 2 hearts) (Fig 1A). In contrast, action potentials from patients with LV hypertrophy had a shape similar to that observed in guinea pig ventricular cells and never showed phase 1 repolarization. The plateau started at the end of the upstroke and was always higher than that observed in the undiseased preparation (Fig 1B). Decay of phase 2 was relatively slow, and the average APD₉₀ was 449±11 ms (n=59, 59 hearts). These action potential patterns suggested that differences in I_to densities may exist between diseased and undiseased tissues from the myocardial region under study.

View larger version (12K): [in this window] [in a new window]   Figure 1. Action potentials recorded in syncytial preparations isolated from superficial (<3 mm deep) subendocardial LV septum from an undiseased heart (A) and from a patient with aortic stenosis and compensated LV hypertrophy (B). Basic cycle length was 2500 ms; temperature, 35°C. A small notch at phase 1 of action potential suggested presence of an Ito in undiseased preparations. In diseased preparations, notch was absent, early plateau phase was higher than in undiseased tissue, and there was a clear action potential prolongation. Resting potential was -79±2 mV (n=4 from 2 hearts) and -82±1 mV (n=59 from 59 hearts) from undiseased and diseased preparations, respectively. APD90 repolarization was: undiseased, 384±23 ms (n=4 from 2 hearts); diseased, 449±11 ms (n=59 from 59 hearts).

Absence of Macroscopic I_to in Subendocardial Hypertrophied Septal Cells
As previously reported,²⁷ a macroscopic I_to, even after superfusion of Ca²⁺ channel blockers, could not be elicited in superficial subendocardial cells of LV septum from patients with aortic stenosis and compensated LV hypertrophy. In a recent series of experiments (n=25 from 8 patients), the application from a -80-mV holding potential of 300-ms voltage-clamp steps to -50 mV up to +60 mV in 10-mV increments every 5 seconds could not evoke any I_to either. Fig 2 shows currents obtained with this protocol after successive application of standard choline chloride Tyrode's solution (A), this solution with 2 mmol/L Co²⁺ added (B), or with 2 mmol/L Co²⁺ and 3 mmol/L 4-AP (C). Traces D represent the subtraction of traces C from those of B, showing that the 4-AP–sensitive current is extremely reduced or absent. This pattern was always found in the 25 cells so studied. Hence, we were prompted to evaluate and compare I_to density in undiseased and diseased myocytes from the superficial subendocardial layers of LV septum.

View larger version (25K): [in this window] [in a new window]   Figure 2. Absence of macroscopic Ito in superficial subendocardial myocytes from hypertrophied septum. Currents were elicited from a -80-mV holding potential by 300-ms voltage-clamp steps to -50 to +60 mV in 10-mV increments (0.2 Hz). A, Currents recorded in control conditions (Na+-free solution). B, Current traces obtained after 3 minutes of 2 mmol/L Co2+ superfusion. C, Currents recorded after 3 minutes' exposure to 2 mmol/L Co2+ and 3 mmol/L 4-AP. D, 4-AP–sensitive currents obtained by subtraction of records in C from records in B. Arrowheads indicate zero current level. Calibrations shown. Cm=125 pF.

Hypertrophy and Heterogeneity Factors of I_to Density
To evaluate I_to density in undiseased and diseased myocytes from superficial subendocardial layers of LV septum, current-voltage relationships for I_to were determined by a voltage-clamp protocol similar to the one mentioned above. Cells were successively superfused with choline chloride solutions first containing 2 mmol/L Co²⁺ and then 3 mmol/L 4-AP. I_peak (positive or negative) and I_late were measured. I_to was defined as the purely transient current component, ie, the difference between I_peak and I_late from current traces elicited in the presence of Co²⁺. Typical current tracings derived from undiseased and diseased cells are shown in Fig 3. In undiseased cells investigated in the Co²⁺-containing Tyrode's solution, outward currents began to emerge at about -20 mV. With increasing depolarization to more positive test potentials, the rate of rise of currents and peak and plateau current amplitudes increased, as previously observed in other regions from human left ventricle.^{16 19 35 36} After reaching its peak within 5 to 25 ms, current decayed with an exponential time course; its inactivation was incomplete at the end of the 300-ms pulse (Fig 3Aa). In contrast, patterns recorded in diseased cells (Fig 3Ba) did not display any rapidly activating or inactivating outward current on depolarization. Instead, a small, time-independent outward current was elicited by depolarizing voltage steps. Peak outward current density at +60 mV reached significantly larger amplitude in undiseased cells (5.88±0.51 pA/pF; range, 4.51 to 6.89 pA/pF; n=4, 2 hearts) than current density measured immediately after the residual capacitive artifact in diseased cells (1.37±0.12 pA/pF; range, 0.12 to 3.06 pA/pF; n=42, 19 hearts) (P=.001) (Table). The density of I_late in undiseased myocytes was 1.64±0.20 pA/pF (range, 1.24 to 2.08 pA/pF) and not very different from that observed in diseased cells (1.29±0.10 pA/pF; range, 0.23 to 2.59 pA/pF) (Table). Cell membrane capacitance was similar in the two groups (undiseased: 107.0±11.0 pF; range, 89 to 156 pF; diseased: 98.2±16.1 pF; range, 48 to 137 pF; P=NS). On washout with a 4-AP–containing solution, slow inward currents were elicited. In undiseased cells (Fig 3Ab), the amplitude of the Co²⁺-sensitive inward current at +10 mV was 4.04±0.35 pA/pF (range, 3.12 to 4.55 pA/pF; n=4, 2 hearts), very similar to that obtained in diseased cells (4.80±0.08 pA/pF; range, 2.18 to 5.47 pA/pF; n=42, 19 hearts) (P=NS) (Fig 3Bb). Hence, under the same experimental conditions, undiseased cells from superficial layers of LV septum were able to evoke both I_to and calcium currents, whereas diseased cells showed only calcium currents.

View larger version (37K): [in this window] [in a new window]   Figure 3. Differences in depolarization-activated outward and inward currents of undiseased (A) and diseased (B) myocytes from superficial subendocardial layers of human LV septum. Holding potential was -70 mV; cells were depolarized in 10-mV increments to test potentials between -50 and +70 mV. Cells were successively superfused by Na+-free Tyrode's solution containing 2 mmol/L Co2+ (row a) and then by Na+-free Tyrode's solution containing 3 mmol/L 4-AP (row b). Membrane capacitance values were 154 pF for representative undiseased cell (A) and 96 pF for diseased cell (B). Arrowheads indicate zero current level. Calibrations shown. Bottom panels show mean current-voltage relations for peak and sustained current at end of test pulse obtained in undiseased myocytes (n=4 from 2 hearts) and in diseased myocytes (n=42 from 19 hearts). Squares indicate Co2+-resistant currents; circles, 4-AP–resistant currents; solid symbols, mean peak density values; open symbols, current densities measured at end of pulse. Ito values are given by differences between solid and open squares in bottom left graph. Values are mean±SEM; in some cases, SEMs are within symbol size.

View this table: [in this window] [in a new window]   Table 1. Ito Densities in Myocytes From Diseased and Undiseased Hearts at +60 mV

Similar voltage-clamp protocols were also applied to diseased myocytes isolated from deep layers of LV septum (3 to 6 mm deep). Fig 4 shows representative current traces and current density–voltage relationships obtained in 4 cells from 2 hearts (mean cell capacitance, 95±12 pF; range, 77 to 142 pF). A rapidly activating I_to that decayed with time to an apparently steady-state plateau could be observed. At +60 mV test potential, I_peak density was 10.51±1.29 pA/pF (range, 7.42 to 13.73 pA/pF), whereas I_late density was 1.91±0.21 pA/pF (range, 1.36 to 2.30 pA/pF). The relative peak-to-plateau current amplitude was larger in these cells than in undiseased superficial subendocardial myocytes (see Fig 3Aa). In these deep subendocardial cells, I_peak density at +60 mV was significantly greater (P=.001) than current density measured immediately after the residual capacitive artifact in superficial subendocardial cells (Table). Conversely, I_late density in deep subendocardial myocytes was not very different from that observed in superficial subendocardial cells (1.29±0.10 pA/pF) (Table). Thus, depending on the depth, macroscopic I_to appeared to be present or absent in myocytes taken from the LV hypertrophied septum.

View larger version (22K): [in this window] [in a new window]   Figure 4. Voltage dependence of Ito in diseased myocytes isolated from deep layers of LV septum (3 to 6 mm deep). Top to bottom, Voltage protocol, original current recordings from a representative cell (Cm=77 pF), and current density–voltage relationships obtained in 4 cells from 2 hearts (mean cell capacitance, 95±12 pF; range, 77 to 142 pF). Arrowhead, zero current level; calibrations shown. Solid squares are mean peak current density values; open squares, mean maintained current at end of pulse. In some cases, SEMs are within symbol size.

Divalent cations have been shown to cause important shifts of activation and inactivation parameters of I_to toward depolarized potentials.³³ Similar behavior after extracellular Cd²⁺ application has also been suggested in human ventricle.¹⁹ Assuming that superficial subendocardial diseased myocytes might be particularly sensitive to this cation-dependent regulatory mechanism, we applied to a subset of cells (n=9, 4 hearts) more depolarized voltage steps up to +110 mV in 10-mV increments from a -70 mV holding potential. No I_to was elicited in these conditions. Provision was also made against a negative shift of I_to availability by delivering, from a -70-mV holding potential, 300-ms prepulses to -140 mV followed by test pulses up to -20 mV with the same negative result (n=9, 5 hearts). Hence, no evidence of the presence of macroscopic I_to could be found in these cells within a large voltage range.

An I_to-Like Current is Unmasked by Extracellular Ca²⁺ Removal
In these cells lacking macroscopic I_to, we then examined the possibility of an extracellular Ca²⁺–dependent regulation of an outward current similar to that previously reported in guinea pig ventricular cells, in which I_to is functionally absent.³⁷ To test this assumption, [Ca²⁺]_o was varied from 0 to 8 mmol/L, and depolarizing test pulses were applied from a -70-mV holding potential to -60 up to +70 mV in 10-mV increments. The pulse protocol was imposed, before and after external Ca²⁺ removal, to superficial subendocardial diseased cells that have been shown to lack I_to. Fig 5 shows a representative example of response patterns obtained in 29 cells from 17 patients. In Ca²⁺-containing choline chloride solution, slow inward currents were elicited in the -30- to +40-mV range, and only outward currents of relatively low amplitude were recorded at potentials positive to the apparent calcium current reversal potential (Fig 5A). Current traces thus obtained were very similar to those previously reported.²⁷ Within the first minute after extracellular Ca²⁺ removal, large time- and voltage-dependent I_o emerged at positive membrane potentials as shown in Fig 5B. The activation threshold was -20 mV. Currents displayed rapid activation, with time to peak ranging from 5 to 10 ms, and then soon decayed toward a sustained component. Current-voltage relations exhibited a steady increase for voltages positive to 0 mV, and at a test potential of +60 mV, the mean peak current density was 30.7±2.6 pA/pF and the mean steady-state current was 13.5±1.6 pA/pF. The dramatic change induced by extracellular Ca²⁺ removal is highlighted by the mean current-voltage plots shown in the bottom panels of Fig 5.

View larger version (29K): [in this window] [in a new window]   Figure 5. Emergence of an Ito-like current induced by extracellular Ca2+ removal in cells from superficial layers of hypertrophied LV septum that have been shown to lack Ito. From a -70-mV holding potential, 1000-ms depolarizing pulses were applied to -60 to +70 mV in 10-mV increments. Currents elicited by such a voltage-pulse protocol were obtained before (A) and 1 minute after (B) extracellular Ca2+ removal. Middle panels show representative example from one cell of response patterns obtained in 29 cells from 17 hearts. B, Inset, initial part of current traces is shown in expanded time scale. Arrowheads, zero current level. Calibrations shown. Bottom panels, Mean current-voltage relations for peak (solid circles) and sustained current present at end of pulse (open circles). In some cases, SEMs are within symbol size. Note large difference in scalings between left and right bottom panels. Cm=123.6 pF.

Is any other divalent cation able to exert similar effects? This matter was addressed by application of 2 mmol/L Co²⁺ on cells that exhibited I_o induced by extracellular Ca²⁺ removal. Fig 6 depicts original recordings from a cell undergoing [Ca²⁺]_o changes and Co²⁺ external superfusion. Before Co²⁺ superfusion, we checked that the cell was responsive to [Ca²⁺]_o increases (Fig 6B). Then, extracellular Ca²⁺ was removed (Fig 6C), and 2 mmol/L Co²⁺-containing solution was subsequently applied (Fig 6D). Clearly, Co²⁺ mimicked the inhibitory effect of Ca²⁺ on I_o. Similar effects were observed in four of five cells tested.

View larger version (32K): [in this window] [in a new window]   Figure 6. Graphs showing sensitivity of Ito-like current to divalent cations. Voltage-pulse protocol is same as used in Figs 3 and 4. All traces are successively obtained in same myocyte (from superficial layers of hypertrophied LV septum). A, Currents are first elicited in control conditions (2 mmol/L Ca2+-containing solution) (top left). B, Then increasing [Ca2+]o to 8 mmol/L induces a marked increase in slow inward current (top right traces). C, In following step (lower left), removal of extracellular Ca2+ suppresses inward calcium current and induces very large time- and voltage-dependent outward currents (note different scalings between top and bottom panels). D, Finally, addition of 2 mmol/L Co2+ (lower right) clearly inhibits outward current induced by extracellular Ca2+ removal. Arrowheads, Zero current level. Calibrations shown. Cm=113.2 pF.

To assess the K⁺ selectivity of I_o, it would have been necessary to estimate its reversal potential from current tails evoked during hyperpolarization to potentials between -30 and -80 mV from a depolarized test potential of +60 mV (using a -70-mV holding potential). Hyperpolarizations were applied 10 or 500 ms after the onset of the +60-mV depolarizing step to study peak- and plateau-related tail currents. Despite careful series resistance compensation, we never could discriminate a tail current from the capacitive transient at any time (data not shown). Deactivation currents were exceedingly fast to be recorded reliably. However, it is well known that I_to can be inhibited by external and internal Cs⁺.^{38 39 40} Hence, assuming an analogous behavior of this current with I_to, we substituted Cs⁺ for K⁺ in both internal and external K⁺ solutions. Both the time-dependent component and the plateau component were strikingly reduced by Cs⁺ application: at +60 mV, mean peak I_o density was about threefold smaller in Cs⁺ than in K⁺ treated cells (K⁺, 30.7±2.6 pA/pF, n=8 cells from 4 hearts; Cs⁺, 9.3±1.8 pA/pF, n=4 from 4 hearts; P<.01). At the end of a 1000-ms test pulse, the maintained current was also significantly reduced in Cs⁺-treated cells compared with the K⁺-treated cells (3.1±0.3 versus 13.5±1.6 pA/pF, respectively; P<.01). In another series of experiments, we looked for a sensitivity of I_o activation to extracellular K⁺ changes, based on the rationale that activation and inactivation parameters of transient outward potassium channels as well as reversal potentials may be influenced by [K⁺]_o.^{41 42} Increasing [K⁺]_o from 4 to 8 mmol/L induced an apparent decrease of the peak component, whereas the plateau component was unchanged during test pulses positive to -60 mV from a -70 mV holding potential. At a +60-mV test potential, I_o density was significantly lower for 8 mmol/L than for 4 mmol/L [K⁺]_o (19.7±2.5 versus 30.7±2.5 pA/pF, respectively; n=8 cells, 4 hearts; P=.02).

The question of whether I_o and I_to would exhibit similar pharmacological properties was then addressed by use of 3 mmol/L 4-AP in the superfusate. In 12 cells from 4 hearts, we consistently observed a lack of effect of 4-AP on I_o (data not shown). External equimolar substitution of tetraethylammonium chloride for choline chloride did not change the amplitude and kinetics of I_o either (n=3).

The decay of the current was analyzed by fitting exponential functions to experimental data. Two exponential terms were needed to describe the time course of inactivation; the best fit was obtained by an equation in the following form: I_o(t)=A_f exp(-t/_f)+A_s exp(-t/_s)+A_c, where I_o(t) is the amplitude of I_o at time t, _f and A_f are the time constant and initial amplitude of the fast component, _s and A_s are the time constant and initial amplitude of the slow component of inactivation, and A_c is the steady-state component. Representative examples of exponential fits are depicted in Fig 7A. There was no obvious voltage dependence in the range of +30 to +70 mV for both fast and slow time constants of inactivation (Fig 7B) (n=6 cells, 6 hearts). At a +60-mV potential, the respective fraction amplitudes of total I_o were 0.41±0.04 for the fast component, 0.18±0.03 for the slow component, and 0.41±0.05 for the noninactivating outward current. To analyze the time course of recovery from inactivation of peak I_o, we used a double-pulse protocol (Fig 7C). Two 500-ms pulses, each to +70 mV with an increasing interpulse interval (10 to 2000 ms), were applied every 10 seconds from a -70-mV holding potential. The magnitude of peak I_o elicited by the test pulse was expressed as a fraction of the peak I_o during the conditioning pulse and plotted against the recovery interval duration. There was no overshoot of the availability of the current at any interpulse interval, and the recovery was slow, with only 93% of the current available after 500 ms. A single exponential fit with an average time constant of 192.0±7.0 ms (n=6 cells, 5 hearts) was required to adequately describe the time course of recovery.

View larger version (24K): [in this window] [in a new window]   Figure 7. Kinetics of Ito-like current. A, Representative fit analysis of current inactivation in response to 1000-ms voltage-clamp pulses applied from +30 to +70 mV (holding potential, -70 mV; frequency, 0.1 Hz). Solid curves, double exponential fits to decay phase of currents. At +30, +40, +50, +60, and +70 mV, best-fit inactivation time constants f and s were (in ms) 25, 394; 23, 137; 29, 253; 28, 195; and 29, 182, respectively. Cm=87 pF. B, Semilogarithmic plots of mean time constants for fast and slow components of Ito-like current showing that inactivation kinetics are voltage insensitive. C, Plot showing mean time course of recovery from inactivation. Inset, Pulse protocol. Recovery had a monoexponential time course in each studied cell (n=6).

These findings suggest that in cells lacking I_to because of a particular physiological state (such as in ventricular guinea pig cells³⁷ ) or an adaptive response to myocardial stress (human septal cells from hypertrophied hearts), extracellular Ca²⁺ removal is able to induce an I_to-like current.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our findings indicate that macroscopic I_to is absent in superficial LV septal layers from hypertrophied hearts, whereas it is clearly present in deep LV septal layers from the same hearts and in superficial LV septal layers from nondiseased hearts. Unexpectedly, in cells lacking I_to, removing Ca²⁺ from the milieu induces a large, rapidly activating and inactivating I_o, which differs from I_to by its much larger magnitude as well as pharmacological and kinetic characteristics.

For action potential recordings, microelectrode techniques on syncytial preparations were chosen rather than current clamp on single cells, because of more stable and higher resting potential levels obtained with this technique in Na⁺-containing solutions. Conversely, no attempt was made to describe gating properties and kinetics of I_to, inasmuch as such features have been previously reported in detail^{16 19 36} and our objective was focused on the heterogeneous modulation of the density of this current.

Action Potential Characteristics
The clear difference in action potential shape between hypertrophied and nonhypertrophied hearts was the presence of a notch between phases 1 and 2 of the action potential in nonhypertrophied tissue. Liu et al¹¹ showed that the action potential characteristics of syncytial preparations are very similar to those observed in individual myocytes of identical regions of the myocardium, leading to the conclusion that different electrical behaviors are to a large extent due to differences in cellular intrinsic properties. Along these lines, we expected that the absence of phase 1 repolarization in superficial subendocardial myocytes from hypertrophied preparations was related to an I_to decrease. Absence of notch, higher voltage levels of the plateau, and longer APDs have recently been observed in human ventricular myocytes from subendocardium that exhibit reduced I_to density.¹⁶ Therefore, the absence of phase 1 repolarization in our multicellular preparations could be indicative of a relatively low density of I_to.

Modulating Factors of I_to Density
I_to density recently investigated in right septal subendocardial myocytes from nonfailing human hearts (6.0 pA/pF at +60 mV) is comparable to our records from undiseased superficial LV septal myocytes (Table).⁴³ Our data are also similar to those gathered by Wettwer et al¹⁹ (4.4±1.1 pA/pF) and Näbauer et al¹⁶ (5 pA/pF) at the same voltage in subendocardial layers of LV anterior wall from donor hearts.

Previous studies have pointed to significant reductions of I_to density during experimental hypertrophy.^{12 21 23 24 25 26} Indeed, in human hearts, cardiac failure seems to bring about similar qualitative changes. In myocytes isolated from the central one third of the LV free wall, I_to density was significantly reduced in failing compared with nonfailing myocardium.³⁵ When comparing the endocardial one third and epicardial one third of myocardial wall, Wettwer et al¹⁹ found a marginally significant decrease in I_to density in endocardial but not epicardial cells in heart failure. In thinner preparations from failing hearts, Näbauer et al¹⁶ showed that I_to density was significantly reduced in subepicardial but not in subendocardial myocytes. Still, clear evidence was provided in both studies of higher densities of I_to in epicardial than in endocardial myocytes in failing and nonfailing hearts. However, as far as compensated hypertrophy is concerned, no inference can be allowed from data collected in patients with terminal heart failure.⁴⁴

Our data provide clear evidence of pronounced alterations in I_to density in superficial layers of LV septum during pressure overload–induced compensated hypertrophy. The lack of macroscopic I_to most likely contributes to the absence of phase 1 and the relatively higher level of early plateau in this region. But the APD₉₀ prolongation cannot be directly accounted for by the absence of this current during the final phase of repolarization, because I_to, when present in human ventricle, exhibits very rapid inactivation kinetics at physiological temperatures.¹⁶ Wettwer et al¹⁹ speculated that the inner layers of myocardial wall may be more susceptible to alterations related to cardiac pathology than the outer tissue. Along these lines, we may suggest that the outflow tract of hypertrophied ventricles can be submitted to high levels of stress responsible for exaggerated adaptive responses leading to a total inhibition of I_to channel synthesis in the surface layers. Conversely, we must emphasize that at very short distance, high densities of I_to are found in deeper layers, and hence, sharp gradients in I_to may occur in hypertrophied human subendocardium: these "deep" I_to densities are even significantly greater than those observed in superficial layers from control septa (P<.03; see Table). Such a clear-cut differential distribution of I_to might be a not unusual phenomenon, because it has recently been demonstrated that steep gradients in I_to also exist physiologically in canine LV epicardium.⁴⁵

Significance of the I_to-Like Current
The emergence of an I_to-like current (I_o) after removal of extracellular Ca²⁺ has already been reported in guinea pig ventricular cells that are known to be lacking I_to.³⁷ Similarities with this current are activation within the same voltage range, half-inactivation voltage at -30 mV (data not shown), and clear inhibition by a divalent cation. Some clues are also indicative of a probable involvement of K⁺ ions as the charge carrier: eg, the sensitivity of the current to external and internal Cs⁺ and to changes in [K⁺]_o. However, I_o was insensitive to 4-AP and external tetraethylammonium chloride.

Several other features differentiate I_o from the classic I_to.^{16 19 40 44} First, peak I_o exhibits a severalfold larger magnitude than peak I_to. Second, the two-exponential decay of I_o inactivation and its monoexponential time course of recovery contrast with the monoexponential decay and the two-exponential time course of recovery usually observed in I_to studies.* Furthermore, I_o was not inducible in human atrial cells already showing I_to (n=6): in these myocytes, extracellular Ca²⁺ removal induced only a 15% to 20% increase of peak I_to at +60 mV because of a shift of the current-voltage relation to more negative potentials reminiscent of that previously reported in rat ventricular myocytes.⁴⁶ Therefore, it seems unlikely that I_o could correspond to the actual I_to unmasked by Ca²⁺ removal. Moreover, since I_o was detected during the early time course of our experiments, we discarded a patch-duration–dependent K⁺ current similar to that described by Martin et al.⁴⁷ Yet insensitivity to 3 mmol/L 4-AP does not exclude an I_to-type channel, because of the very wide range of sensitivity to this drug among the I_to-type family of currents.⁴⁸ Indeed, hypertrophy-induced molecular changes of the I_to channel protein may be the underlying cause of the emerging I_o, without excluding the role of a specific channel inhibited by extracellular calcium.

Physiological Implications
The absence of a macroscopic I_to in superficial but not deep subendocardial LV septal layers from hypertrophied hearts may have a significant bearing on the electrical behavior of human ventricle. The resulting increase of regional heterogeneity enhances differences in restitution and rate dependence of APD and refractoriness in these tissue layers. Then a marked dispersion of repolarization within the septum may provide the electrophysiological substrate for the genesis of reentrant arrhythmias.¹ In addition, such a situation can induce pronounced differences in the responsiveness to drugs and ischemia and thus represent a potentially deleterious factor.⁴⁹ Conversely, the significance of the I_to-like current appears unclear, but its possible involvement in not yet defined pathophysiological circumstances cannot be ruled out.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine APD = action potential duration APD90 = action potential duration at 90% of repolarization Cm = membrane capacitance Ilate = current at end of depolarizing pulses Io = outward current Ipeak = peak current Ito = transient outward current LV = left ventricular

	Footnotes

 

Received September 23, 1996; accepted December 11, 1996.

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