Transmural heterogeneity of action potentials and Ito1 in myocytes ...

Transmural heterogeneity of action potentials and Ito1 in myocytes isolated from the human right ventricle GUI-RONG LI,1 JIANLIN FENG,1 LIXIA YUE,1 AND MICHEL CARRIER2 of Medicine and 2Department of Surgery, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada H1T 1C8

1Department

Li, Gui-Rong, Jianlin Feng, Lixia Yue, and Michel Carrier. Transmural heterogeneity of action potentials and Ito1 in myocytes isolated from the human right ventricle. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H369–H377, 1998.— Limited information is available about transmural heterogeneity in cardiac electrophysiology in man. The present study was designed to evaluate heterogeneity of cardiac action potential (AP), transient outward K1 current (Ito1 ) and inwardly rectifying K1 current (IK1 ) in human right ventricle. AP and membrane currents were recorded using whole cell current- and voltage-clamp techniques in myocytes isolated from subepicardial, midmyocardial, and subendocardial layers of the right ventricle of explanted failing human hearts. AP morphology differed among the regional cell types. AP duration (APD) at 0.5–2 Hz was longer in midmyocardial cells (M cells) than in subepicardial and subendocardial cells. At room temperature, observed Ito1, on step to 160 mV, was significantly greater in subepicardial (6.9 6 0.8 pA/pF) and M cells (6.0 6 1.1 pA/pF) than in subendocardial cells (2.2 6 0.7 pA/pF, P , 0.01). Slower recovery of Ito1 was observed in subendocardial cells. The half-inactivation voltage of Ito1 was more negative in subendocardial cells than in M and subepicardial cells. At 36°C, the density of Ito1 increased, the time-dependent inactivation and reactivation accelerated, and the frequency-dependent reduction attenuated in all regional cell types. No significant difference was observed in IK1 density among the regional cell types. The results indicate that M cells in humans, as in canines, show the greatest APD and that a gradient of Ito1 density is present in the transmural ventricular wall. Therefore, the human right ventricle shows significant transmural heterogeneity in AP morphology and Ito1 properties. electrophysiology; ionic channels

in cardiac electrophysiology has been demonstrated in several species (5, 8, 13, 15, 35) and is considered to be an important factor for understanding electrical properties of hearts and arrhythmic mechanisms (3, 4). Transmural electrical differences were first demonstrated by the recording of transmembrane action potentials in ventricular tissues (3, 5, 15, 28). The studies (4, 5, 38) have provided the evidence for three functionally different cell types in the canine ventricular wall with a distinct electrophysiological profile. These cell types include endocardial cells, epicardial cells, and a subpopulation of midmyocardial layer cells (M cells) in the deep subepicardial layer. The M cells have action potential durations (APD) longer than and electrophysiological features intermediate between those of myocardial and conducting cells (4, 5, 38), with pharmacological responsiveness different from that of either epicardial or endocardial cells (9, 10, 29, 33). The differences in action potential characteristics are mainly due to different

TRANSMURAL HETEROGENEITY

intrinsic electrophysiological properties (9, 10, 29, 33, 38). It has been shown that a 4-aminopyridinesensitive transient outward K1 current (Ito1 ) contributes to the difference in early phase of the action potential in the ventricular wall (5, 28, 31), whereas the lower density in the slow component of delayed rectifier K1 current (IKs ) has been thought to be related to longer APD in M cells (17, 30). The differences in action potential morphology and Ito1 were described in subepicardial and subendocardial cells isolated from the left ventricle of human hearts (6, 34, 41). M cells were not studied with regard to properties of the action potential and Ito1 in these reports. Drouin et al. (10) reported that heterogeneity in the action potential and M cells were present in the human left ventricle, and the M cell shows features similar to those in the canine ventricle, namely longer APD and greater maximal rate of the increase in action potential upstroke (10). However, whether transmural heterogeneity in the action potential and Ito1 properties is present in the human right ventricle is unknown. The purposes of the present study were to evaluate 1) transmural heterogeneity in action potentials, Ito1, and inwardly rectifying K1 current (IK1 ), and 2) temperature dependence of Ito1 in density and kinetics in right ventricular myocytes isolated from subepicardial, midmyocardial, and subendocardial layers of explanted human hearts. MATERIALS AND METHODS

Myocyte preparation. Right ventricular tissues from explanted hearts were obtained at the time of heart transplantation in patients. The ventricular cells were isolated using a procedure described previously (26). Briefly, all hearts were initially placed in cold (4°C) oxygenated Krebs solution and then transferred to cardioplegic solution for dissection and coronary artery cannulation. A portion of the free transmural wall of the right ventricle (,20 3 40 mm) was removed along with the coronary artery branch irrigating it, with dissection and arterial cannulation completed within 30 min of excision of the heart. The free wall was perfused with oxygenated, nominally Ca21-free Tyrode solution for 20–30 min, and the solution was then changed to one containing 200–300 U/ml collagenase (CLS II, Worthington Biochemical, Freehold, NJ) for 60–100 min. Regional myocytes were separated from the digested tissue. Subendocardial and subepicardial cells were taken from the endocardial and epicardial surfaces (,1 mm thick), whereas M cells were dissociated from the midmyocardial layer. The cells were placed in a high-K1 storage solution (see Solutions) and gently triturated with a Pasteur pipette. Isolated myocytes were kept in the medium at least 1 h (at room temperature) before use. Cells used in the present study were from seven hearts. The underlying heart disease was congestive cardiomyopathy in five cases and heart failure due to aortic valve disease in

0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society

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TRANSMURAL HETEROGENEITY IN AP AND ITO1

two cases. Subsequent examination of the right ventricle by a cardiac pathologist revealed it to be microscopically normal in four cases, to show minor extramyocardial abnormalities consisting of fatty infiltration in one case and subendocardial fibrosis in another, and to show cellular hypertrophy in the final case. A small aliquot of the solution containing the isolated cells was placed in an open perfusion chamber (1 ml) mounted on the stage of an inverted microscope. Myocytes were allowed to adhere to the bottom of the dish for 5–10 min and were then superfused at 2–3 ml/min with Tyrode solution. Only quiescent, rod-shaped cells showing clear cross-striations were used. Solutions. The Tyrode solution contained (in mmol/l) 136 NaCl, 5.4 KCl, 1 MgCl2, 2 CaCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH. The high-K1 storage medium contained (in mmol/l) 20 KCl, 10 KH2PO4, 10 glucose, 70 K-glutamate, 10 b-hydroxybutyric acid, 10 taurine, 5 EGTA, and 10 mannitol as well as 0.1% albumin, with pH adjusted to 7.2 with KOH. The pipette solution contained (in mmol/l) 20 KCl, 110 K-aspartate, 1 MgCl2, 10 HEPES, 5 EGTA (0.05 for the recording of action potentials), 0.1 GTP, 5 Na2-phosphocreatine, and 5 Mg2-ATP, with pH adjusted to 7.2 with KOH. For Ito1 determination, BaCl2 (0.5 mmol/l) was used to inhibit IK1, and CdCl2 (0.2 mmol/l) was used to block Ca21 current (ICa ). Ca21-dependent transient outward Cl2 current (ICl,Ca or Ito2 ) was inhibited by 5 mmol/l EGTA in the pipette solution and by the addition of Cd21 to the external solution. The experiments were conducted at 36°C and/or room temperature (specified). Data acquisition and analysis. The whole cell patch-clamp technique was used. Borosilicate glass electrodes (1.0-mm OD) were pulled with a Brown-Flaming puller (model P-87) and had tip resistances of 2–3 MV when filled with pipette solution. The tip potentials were compensated before the pipette touched the cell. A gigaseal (.10 GV) was obtained, and the cell membrane was ruptured by gentle suction to establish the whole cell configuration. Data were acquired with the use of an Axopatch 200A and/or 200B amplifier (Axon Instruments, Foster City, CA). Command pulses were generated by a 12-bit digital-to-analog converter controlled by pCLAMP software (Axon Instruments). Recordings of the action potential and the current were low-pass filtered at 2 kHz and stored on the hard disk of an IBM-compatible computer. Cell capacitance (values are shown in RESULTS ) was calculated by integrating the area of the capacitive response to a 5-mV hyperpolarizing step from a holding potential of 260 mV (in the presence of 0.5 mmol/l BaCl2 ) divided by 5 mV. The series resistance (Rs ) was electrically compensated to minimize the duration of the capacitive transient. Rs along the clamp circuit was estimated by dividing the time constant obtained by fitting the decay of the capacitive transient by the cell membrane capacitance. Before Rs compensation, decay of the capacitance was expressed by a single exponential with a time constant of 1,107 6 108 µs and an Rs of 6.7 6 0.5 MV. After compensation, the values were reduced to 336 6 23 µs and 2.1 6 0.3 MV, respectively. Action potentials were recorded in current-clamp mode. Recorded resting potentials were corrected for liquid junction potentials. To calculate the junction potential, pipettes filled with solution were immersed into a solution identical to the pipette solution and subsequently immersed into the bath solution used for action potential recording. The potential difference recorded was the junction potential of the pipette. The average junction potential (10.5 6 0.3 mV) of 14 pipettes was subtracted from the resting membrane potential.

The temperature coefficient (Q10 ) (20) for Ito1 density was calculated by using the equation Q10 5 1 1 10(A2 2 A1 )/[A1 (T2 2 T1 )], where A1 and A2 are Ito1 density at different temperatures T1 and T2, whereas Q10 for Ito1 inactivation and reactivation time constants was calculated with the equation Q10 5 exp 5[10/(T2 2 T1 )] ln (t1/t2 )6, where t1 and t2 are the time constants obtained at T1 and T2, respectively. Nonlinear curve-fitting programs [Clampfit in pCLAMP 6 (Axon Instruments) or SigmaPlot (Jandel Scientific, Rafael, CA)] was used to perform curve-fitting procedures. Results are presented as means 6 SE. Paired and unpaired Student’s t-tests were used as appropriate to evaluate the statistical significance of differences between two group means, and ANOVA was used for multiple groups. Values of P , 0.05 were considered to indicate statistical significance. RESULTS

Cell capacitance. The average membrane capacitance of human right ventricular myocytes was 162 6 12 pF in subepicardial cells (n 5 41), 167 6 16 pF in midmyocardial cells (n 5 58), and 158 6 14 pF in subendocardial cells (n 5 57). No significant difference was observed in membrane capacitance among the regional cell types. The currents described below as the current density (pA/pF) were obtained relative to individual cell capacitance for control of variation in cell size. Transmural heterogeneity in action potentials. Action potentials were determined with a train of 15 depolarization current pulses (2-ms duration) with frequencies of 0.5, 1, and 2 Hz (60-s interval between trains) in current-clamp mode. Figure 1 shows typical action potentials recorded from subepicardial, midmyocardial, and subendocardial cells at the 15th pulse. The subepicardial cells exhibited a large phase 1 spike, followed by a notch of the action potential similar to that previously reported in canine ventricles and that is considered to be related to the inactivation of Ito1 (4, 5, 31). The midmyocardial cells displayed longer APD (426 6 29 ms, at 1 Hz) than subepicardial (298 6 17 ms) and subendocardial cells (281 6 27 ms) and a large phase 1 magnitude, but not a notch of the action potential. Subendocardial cells did not show an apparent phase 1 spike or a notch of the action potential. No significant difference was observed in the resting potential among the regional cell types: 282 6 2 mV for subepicardial cells (n 5 21); 283 6 3 mV for midmyocardial cells (n 5 31); and 281 6 3 mV for subendocardial cells (n 5 20). The mean values of rate-dependent changes in APD are shown in Fig. 2. At 50% (APD50 ) and 90% repolarization (APD90 ), APD decreased with the increase of stimulation frequency. Both APD50 and APD90 in midmyocardial cells were longer (P , 0.01, n 5 21) than in subepicardial (n 5 19) and subendocardial cells (n 5 15) at all frequencies. At 1 Hz, APD50 and APD90 were 198 6 33 and 263 6 33 ms, 314 6 23 and 390 6 30 ms, and 227 6 15 and 271 6 13 ms, respectively, in subendocardial, midmyocardial, and subepicardial cells. Inwardly rectifying K1 current. The transmembrane current was determined using 300-ms steps from a holding potential of 240 mV to between 2120 and 220 mV in increments of 10 mV (Fig. 3B, inset) every 2 s.


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Fig. 2. Mean values of rate dependence of action potential duration (APD) in regional cell types. APD at 50% repolarization (APD50; A) and at 90% repolarization (APD90; B) showed rate-dependent decrease as frequency increased. APD50 and APD90 were significantly longer in M cells (n 5 21) cells than in Endo (n 5 15) and Epi cells (n 5 19) at all frequencies. Data are means 6 SE. ** P , 0.01 vs. Endo or Epi cells.

(n 5 19). No significant difference in IK1 density was observed in inward (at 290 mV) and outward (at 250 mV) components among the regional cell types [P 5 not significant (NS)].

Fig. 1. Representative action potentials recorded from regional cell types. A: subepicardial (Epi) cell. B: midmyocardial (M) cell. C: subendocardial (Endo) cell. Stimulus frequencies were 0.5, 1, and 2 Hz.

Figure 3, A and B, displays representative recordings from a subepicardial cell in the absence and presence of 0.5 mmol/l Ba21. The membrane current was fully suppressed by the addition of Ba21 to the superfusion solution, indicating that the current elicited by the voltage protocol shown is IK1. The peak and steadystate currents of IK1 were measured from zero current to the current peak and steady-state level at the end of the voltage steps. Figure 3C shows current-voltage (I-V) relationships of the average values of IK1 density (between 2100 and 220 mV). Steady-state IK1 densities at 290 and 250 mV were 210 6 1.7 and 1.5 6 0.3 pA/pF for subepicardial cells (n 5 16), 28.3 6 0.8 and 1.8 6 0.3 pA/pF for midmyocardial cells (n 5 23), and 28.2 6 0.7 and 1.8 6 0.4 pA/pF for subendocardial cells

Fig. 3. Inwardly rectifying K1 current (IK1 ). A: representative IK1 tracings recorded from an Epi myocyte in response to 300-ms test potentials (TP) from 2120 to 220 mV from a holding potential of 240 mV (inset, B). B: IK1 was highly inhibited by addition of 0.5 mmol/l Ba21 to external solution. C: current-voltage (I-V) relationships for peak (left) and steady-state (right) current densities of IK1 in Epi, M, and Endo cells. There was no statistical difference in IK1 among regional cell types. Data are means 6 SE.

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Heterogeneity in density of Ito1. Because temperature may significantly affect the amplitude and kinetics of membrane currents (1, 20, 27), Ito1 was determined at room temperature and at 36°C with a 30-ms step to 240 mV, followed by 300-ms depolarizing steps between 230 and 160 mV in increments of 10 mV every 10 s from a holding potential of 280 mV (Fig. 4C, inset). To directly analyze the effect of temperature, we studied Ito1 at room temperature (23°C) and then repeated the measurements at 36°C in the same cells to determine the temperature dependence of changes in Ito1 amplitude and kinetics in all regional cell types. Figure 4 displays these values of amplitude and density of Ito1 at room temperature and 36°C. Representative recordings from subepicardial, midmyocardial, and subendocardial cells are shown at room temperature (Fig. 4, A–C, a) and at 36°C (Fig. 4, A–C, b) in the same cells. Ito1 amplitude clearly increased at 36°C in all regional cell types. The amplitude of Ito1 was measured from the current peak to the steady-state level at the end of the voltage steps, and I-V relationships of Ito1 density are shown in Fig. 4, A–C, c. The density of Ito1 significantly augmented at 36°C in subepicardial, midmyocardial, and subendocardial cells. At 160 mV, Ito1 (at room temperature and 36°C) was 6.9 6 0.8 and 9.8 6 1.0 pA/pF in subepicardial cells (increased by

Fig. 4. Temperature dependence of Ito1. Representative tracings recorded from an Epi (A), M (B), and Endo cell (C) at room temperature (RT; a) and at 36°C (b). A–C, c: I-V relationships of Ito1 density in Epi (n 5 10), M (n 5 11), and Endo cells (n 5 9), respectively. Data are means 6 SE. * P , 0.05, ** P , 0.01 vs. RT.

42.3 6 2.1%, n 5 10, P , 0.01; Q10 5 1.31 6 0.09), 6.0 6 1.1 and 8.6 6 1.2 pA/pF in midmyocardial cells (increased by 43.5 6 2.5%, n 5 11, P , 0.01; Q10 5 1.34 6 0.11), and 2.2 6 0.7 and 3.3 6 0.8 pA/pF in subendocardial cells (increased by 46.4 6 1.5%, n 5 9, P , 0.01; Q10 5 1.37 6 0.07). Figure 4 also shows that the gradient of Ito1 density is present in myocytes from the human transmural right ventricular wall. The densities of Ito1 were much smaller at room temperature and 36°C in subendocardial cells than in midmyocardial or subepicardial cells (P , 0.05 or P , 0.01, between 110 and 160 mV) and were slightly smaller in midmyocardial cells than in subepicardial cells (P 5 NS). Voltage-dependent inactivation and activation of Ito1. The voltage dependence of Ito1 inactivation was determined at room temperature and 36°C with 1,000-ms conditioning potentials between 290 and 130 mV in increments of 10 mV every 10 s, and then Ito1 was recorded during a 300-ms test pulse to 150 mV. Figure 5A shows the protocol and representative recordings (at room temperature) used to assess Ito1 inactivation. The inactivation variable (denoted as B`, see Ref. 36) was determined by normalizing Ito1 at a given prepulse potential with the maximum Ito1 at 290 mV prepulse. Figure 5B shows the results obtained from the analyses


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The voltage dependence of Ito1 was not altered by the increase of bath temperature. At 36°C, the average V0.5 was 221.2 6 1.1, 225.1 6 1.3, and 232.9 6 1.5 mV, and the average slope factor was 7.3 6 0.4, 7.4 6 0.5, and 8.0 6 0.5 mV, in subepicardial, midmyocardial, and subendocardial cells, respectively (P 5 NS vs. results at room temperature). The voltage dependence of Ito1 activation was determined by calculating an activation variable (denoted as A`, see Ref. 36) with data from Fig. 4, c (measured reversal potential about 268 mV, data not shown). The results are displayed in Fig. 5B. Normalized mean data at room temperature are represented by the symbols, and the curves are Boltzmann fits. No difference in V0.5 and slope factor was observed among regional cell types (Table 1). The V0.5 and slope factor for Ito1 activation were not changed by increasing bath temperature, and similar values were obtained at 36°C. Time-dependent inactivation of Ito1. The time dependence of Ito1 inactivation was studied with 300-ms depolarizing pulses in regional cell types at both room temperature and 36°C. The time-dependent inactivation of Ito1 was reported to be a single exponential in

Fig. 5. Voltage-dependent activation and inactivation of Ito1 in regional cell types. A: representative recordings used to determine voltage dependence of Ito1 inactivation with protocol shown in inset. B: mean voltage-dependent activation and inactivation relationships for Ito1 at RT. Data were fit to Boltzmann relations of the form A` 5 51 1 exp [(V0.5 2 V)/K]621 and B` 5 51 1 exp [(V 2 V0.5 )/K]621 for activation and inactivation, respectively, where V is test or conditioning potential (CP), V0.5 is potential for half-maximal activation or inactivation, and K is a slope factor. Values of A` and B` were calculated as described in text. Data are means 6 SE.

of voltage-dependent inactivation of Ito1 in subepicardial, midmyocardial, and subendocardial cells at room temperature. Mean values are represented by the symbols, and the curves are best-fit Boltzmann functions. As Table 1 shows, the half-inactivation voltage (V0.5 ) was more negative in subendocardial cells than in subepicardial and midmyocardial cells (P , 0.05), and the average slope factor was similar among the regional cell types. Table 1. Parameters of Ito1 voltage-dependent inactivation and activation Inactivation

Activation

Cell Types

n

V0.5 , mV

SP, mV

V0.5 , mV

SP, mV

Endo M Epi

10 11 15

235.2 6 2.3* 226.6 6 1.1 222.5 6 0.7

7.9 6 0.7 9.3 6 0.5 9.1 6 0.3

12.3 6 3.7 12.6 6 3.1 11.5 6 2.7

11.8 6 1.9 11.6 6 1.7 11.2 6 1.2

Values are means 6 SE; n 5 no. of subendocardial (Endo), midmyocardial (M), or subepicardial cells (Epi). Ito1 , transient outward K1 current; V0.5 , half-inactivation or -activation voltage; SP, slope factor. * P , 0.05 vs. M or Epi cells.

Fig. 6. Time-dependent inactivation of Ito1 at RT and 36°C. A: representative current tracings recorded from an M cell at RT and 36°C with protocol shown in inset. Tracings were fitted by a biexponential function (curves shown as solid lines, points are raw data). t1 and t2, Ito1 inactivation time constants. B: voltage dependence of t1 and t2. Both t1 and t2 were significantly smaller at 36°C than at RT in Epi (n 5 10), M (n 5 8), and Endo cells (n 5 8). Data are means 6 SE. ** P , 0.01 vs. RT.

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Table 2. Inactivation time constants of Ito1 and Q10 values RT

36°C

Q10

Cell Types

n

t1 , ms

t2 , ms

t1 , ms

t2 , ms

t1

t2

Endo M Epi

8 8 10

16.9 6 3 15.9 6 4 17.5 6 3

57.9 6 7.8 56.3 6 8.1 60.3 6 8.5

6.4 6 0.6* 5.9 6 0.7* 6.2 6 0.9*

31.9 6 8* 37.9 6 9* 33.7 6 9*

1.61 6 0.06 1.71 6 0.05 1.55 6 0.06

1.61 6 0.05 1.30 6 0.06 1.34 6 0.05

Values are means 6 SE. RT, room temperature; Q10 , temperature coefficient; t1 and t2 , fast and slow time constants of Ito1 at 160 mV. * P , 0.01 vs. RT.

human ventricular myocytes (21, 34, 41). However, we found that Ito1 tracings from 130 to 160 mV were not well fitted by a monoexponential equation in most of the cells studied at room temperature, and at 36°C, Ito1 tracings were only best fitted by a biexponential equation. Therefore, we applied the biexponential equation to fit Ito1 tracings to evaluate the temperature-dependent inactivation of Ito1. Figure 6A shows typical recordings obtained from a midmyocardial cell during a 300-ms voltage step to 140 mV. The raw data were best fitted by a biexponential function with the time constants t1 and t2: 16.5 and 57.2 ms at room temperature, and 8.1 and 32.5 ms at 36°C. The time-dependent inactivation of Ito1 was faster at 36°C than at room temperature, and Q10 was 1.72 for t1 and 1.54 for t2 in this cell. Figure 6B displays the average values of Ito1 inactivation time constants at room temperature and 36°C in the regional cell types. No significant regional and voltage-dependent differences were observed in time constants of Ito1 inactivation. However, both t1 and t2 were much smaller at 36°C (P , 0.01), indicating that the time-dependent inactivation of Ito1 is substantially accelerated by the

Fig. 7. Reactivation of Ito1 in regional cell types at RT and 36°C. A: representative tracings recorded from an M cell at RT and 36°C with protocol shown in inset. P1 and P2, identical 300-ms pulses delivered at varying P1-P2 intervals (Dt). B: reactivation curves for Ito1 at RT and 36°C were best fitted by a biexponential function in Epi (n 5 10), M (n 5 8), and Endo cells (n 5 7). At RT, rapid and slow reactivation time constants (t1 and t2 ) were larger (109.9 and 806.7 ms) in Endo cells than in M (47.2 and 361.3 ms) and Epi cells (48.3 and 483.9 ms), and slower recovery was observed in Endo cells. Physiological body temperature (36°C) clearly accelerated Ito1 recovery in all regional cell types.

increase of bath temperature. Inactivation time constants of Ito1 at 160 mV and their Q10 values are shown in Table 2. The time dependence of Ito1 reactivation was studied at room temperature and 36°C with a paired-pulse protocol illustrated in Fig. 7A (inset). Figure 7A displays representative tracings recorded from a midmyocardial cell at room temperature and 36°C. Identical 300-ms pulses (P1 and P2 ) to 150 mV from a holding potential of 280 mV were delivered every 10 s, with varying P1-P2 intervals. The current during P2 relative to the current during P1 was determined as a function of the P1-P2 reactivation interval. Curves in Fig. 7B show nonlinear curve fits to averaged data in subepicardial, midmyocardial, and subendocardial cells at room temperature and 36°C. Ito1 was not completely recovered during the interval observed. Curves at both room temperature and 36°C were well fitted by a biexponential function. As Table 3 shows, the reactivation time constants t1 and t2 of Ito1 were larger in subendocardial cells than in subepicardial and midmyocardial cells (P , 0.01), indicating slower recovery of Ito1 from inactivation. At 36°C, t1 and t2 were smaller in all

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Table 3. Reactivation time constants of Ito1 and Q10 values RT

36°C

Q10

Cell Types

n

t1 , ms

t2 , ms

t1 , ms

t2 , ms

t1

t2

Endo M Epi

8 10 12

110 6 17† 49 6 12 47 6 9

812 6 87† 484 6 57 369 6 22

34 6 5*† 20 6 3* 12 6 2*

490 6 31* 254 6 24* 229 6 23*

2.42 6 0.12 2.21 6 0.11 2.78 6 0.13

1.45 6 0.16 1.61 6 0.14 1.43 6 0.13

Values are means 6 SE. * P , 0.01 vs. RT; † P , 0.01 vs. M or Epi cells.

regional cells (P , 0.01). The results indicate that an increase in bath temperature accelerates Ito1 reactivation. Q10 values for t1 and t2 were similar in subendocardial, midmyocardial, and subepicardial cells (P 5 NS among the regional cell types). Frequency-dependent reduction of Ito1. Because Ito1 reactivation showed significant time dependence, frequency-dependent reduction of Ito1 might be expected at physiologically relevant frequencies. Changes are shown in Ito1 with repeated pulsing at 0.1, 0.5, 1, 2, and 3 Hz by a 15-pulse train (60-s interval between trains) (Fig. 8B, inset) at room temperature and 36°C in the same regional cells (Fig. 8, A and B, respectively). Steadystate current (at 15th pulse) was normalized to values at 1st pulse at each frequency. Compared with that at 0.1 Hz, significant Ito1 frequency-dependent reduction was observed at room temperature from 0.5 to 3 Hz in subendocardial (n 5 7) cells and from 1 to 3 Hz in midmyocardial (n 5 7) and subepicardial (n 5 8) cells. Ito1 significantly decreased at 1 Hz to 80 6 8, 90 6 2, and 96 6 2% in subendocardial, midmyocardial, and subepicardial cells, respectively (P , 0.05 or P , 0.01). The frequency-dependent reduction of Ito1 was greatly attenuated by the increase of bath temperature (to 36°C; Fig. 8B, P , 0.05 or P , 0.01 vs. results at room temperature), and Ito1 reduced at 1 Hz to only 90 6 10, 99 6 1, and 99.5 6 1% in subendocardial (P , 0.01), midmyocardial (P 5 NS), and subepicardial cells (P 5 NS), respectively.

explanted human hearts. M cells in the human right ventricle showed longer APD. Drouin et al. (10) have recently reported evidence for the presence of M cells in human left ventricles. The M cell in the human left ventricle showed characteristics similar to those in canine ventricles (4, 5, 38), namely longer APD and greater maximal rate of rise of upstroke of the action potential. However, a notch of the action potential was not clear in the M cell of the human left ventricle (10), which is consistent with our observations in the right ventricle. We and Drouin et al. (10) found that APD is still longer in M cells than in epicardial and endocardial cells at physiological heart rates. We did not find a significant difference in IK1 among the three regional cell types. In most of the studies on Ito1 in human ventricles, experiments have been conducted at room tempera-

DISCUSSION

In the present study, we demonstrated that 1) transmural heterogeneity of the action potential morphology exists in the human right ventricle, 2) a gradient of Ito1 density and differences in Ito1 kinetics are present in cells isolated from the transmural right ventricular wall, and 3) the density and time-dependent kinetics of Ito1 are significantly altered by increasing the bath temperature from room temperature to 36°C. Comparison with previously published studies of action potential morphology and Ito1 in human ventricular cells. Two groups have previously described the differences of action potential shape (34) and/or Ito1 (6, 34, 41) in human ventricular subepicardial and subendocardial cells. Our study differs from these in that we characterized the action potential and Ito1 in cells from three regional zones, the subendocardium, midmyocardium, and subepicardium of the human right ventricle. We provided evidence that heterogeneity in action potential morphology and a gradient of Ito1 were present in myocytes isolated from the right ventricle of

Fig. 8. Frequency dependence of Ito1 at RT (A) and 36°C (B) in regional cell types. Results for each cell and each frequency at 15th pulse were normalized to current during 1st pulse. Data are means 6 SE in Epi (n 5 8), M (n 5 7), and Endo cells (n 5 7). * P , 0.05, ** P , 0.01 vs. 0.1 Hz; § P , 0.01 vs. RT.

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ture. We (27) and others (1, 20) have found that the density and kinetics of membrane currents are dependent on bath temperature. Therefore, we determined temperature dependence of Ito1 density and kinetics and then evaluated Q10. By increasing the bath temperature to 36°C, the density of Ito1 was increased. The time-dependent inactivation and reactivation were substantially accelerated, and the frequency-dependent inactivation was attenuated in all regional cell types. However, the voltage-dependent inactivation and activation of Ito1 were not affected by the change of bath temperature. Information about Q10 values for Ito1 is not available to compare with the present study. Q10 values (1.3–1.37) for Ito1 amplitude were smaller than those of other currents (ICa, IK1, and IK, see Refs. 1, 20). The densities (at 160 mV) of Ito1 in the present study were 2.2 6 0.7, 6.0 6 1.1, and 6.9 6 0.8 pA/pF in subendocardial, midmyocardial, and subepicardial cells, respectively, at room temperature. The data from our observation in subepicardial and subendocardial cells are close to those reported by Wettwer et al. (41). They reported that the densities of Ito1 were 2.3 6 0.3 pA/pF (at 160 mV) for subendocardial cells and 7.9 6 0.7 pA/pF for subepicardial cells in human normal left ventricles. However, a higher density of Ito1 in subepicardial cells was reported by Na¨bauer et al. (34) that is probably due to different experimental conditions and/or tissue types. Our observation is consistent with previous reports (34, 41) that Ito1 recovery is slower in subendocardial cells than in subepicardial cells. Significance of our studies for electrophysiology of human heart. The present study provides evidence that the heterogeneity of the action potential and Ito1 exists in the transmural wall of the human right ventricle. The action potential morphology in M cells is similar to that recorded by Drouin et al. (10) in the human left ventricular tissue. Similarly, M cells have been reported in the subendocardium in the anterior wall and septum and in the deep subepicardium in the posterolateral walls of canine hearts (4, 5, 39), which has been considered to be related to the generation of the electrocardiogram U wave (4). The findings from the present study further support the hypothesis that M cells contribute importantly to the manifestation of the U wave or highly bifurcated T waves on the clinical electrocardiogram (4, 10, 23). The present study has demonstrated that the phase 1 variation of the action potential is associated with the gradient of Ito1 in cells from the transmural ventricular wall. Ito1 shows less frequency-dependent reduction at 36°C. The property is similar to that of Ito1 in human atrial myocytes (2, 14), suggesting that Ito1 plays an important role in action potential repolarization of the human myocardium at normal heart rates. It has been shown that Ito1 plays an important role in the mammalian myocardium (5, 7, 8, 15, 16, 28, 41). Liu et al. (31) have recently demonstrated that the distinguished electrical behavior and pharmacological responses are related to the transmural heterogeneity of Ito1 in the canine ventricle. The greater density of Ito1 was found to contribute to the high sensitivity of

epicardial myocardium to electrical depression during ischemia (33). Several antiarrhythmic agents (11, 19, 22) and Ca21 antagonists (18, 24) have been reported to block Ito1. It has been reported that Ito1 might be modulated by adrenergic stimulation (12), atrionatriuretic factor (25), metabolic inhibitor, and oxidant stress (35). Also, the density of Ito1 might be altered by pathological conditions, including cardiac hypertrophy (40) or failure (32) and hyperthyroidism (37). All of the above would have immediate clinical implications, because the gradient of Ito1 is present in the transmural ventricular wall of human hearts. Potential limitations. We studied right ventricular cells from patients with severe left ventricular failure. Although the cells used in the present study were from right ventricles that were assessed by expert pathological microscopic examination to have relatively mild or no changes, and the characteristics of action potentials of our cells were similar to those previously reported by Drouin et al. (10) in the normal human left ventricular tissue, we cannot exclude the possibility that our results were influenced by a relatively mild change in cardiac tissue, such as fatty infiltration, subendocardial fibrosis, or cellular hypertrophy. In conclusion, this study demonstrates that M cells are present in the right ventricle in humans and that a gradient of Ito1 exists in cells across the human right ventricular wall from the endocardium to the epicardium. Ito1 in subendocardial cells is smaller, inactivates more negatively, and recovers more slowly than in midmyocardial and subepicardial cells. Therefore, the human heart shows significant transmural heterogeneity in the action potential and Ito1 properties, which points to an important clinical relevance in human cardiac electrophysiology. We thank Dr. Alvin Shrier for review, constructive suggestions, and discussion concerning the manuscript; Dr. Tack Ki Leung for expert pathological examination of explanted human hearts; and Haiying Sun for data analysis and technical support. The study was supported by the Quebec Heart Foundation and Fonds de la Recherche en Sante´ du Que´bec (FRSQ). G.-R. Li was a research scholar of FRSQ. Address for reprint requests: G.-R. Li, Research Center, Montreal Heart Institute, 5000 Belanger St. East, Montreal, Quebec, Canada H1T 1C8. Received 15 December 1997; accepted in final form 20 April 1998. REFERENCES 1. Allen, T. J. A. Temperature dependence of L-type calcium channel current in single guinea pig ventricular myocytes. J. Cardiovasc. Electrophysiol. 7: 307–321, 1996. 2. Amos, G. J., E. Wettwer, F. Metzger, Q. Li, H. M. Himmel, and U. Ravens. Differences between outward current of human atrial and subepicardial ventricular myocytes. J. Physiol. (Lond.) 491: 31–50, 1996. 3. Antzelevitch, C., S. H. Litovsky, and A. Lukas. Ventricular epicardium vs. endocardium: electrophysiology and pharmacology. In: Cardiac Electrophysiology: From Cell to Bedside, edited by D. Zipes and J. Jalife. Philadelphia, PA: Saunders, 1990, p. 385–395. 4. Antzelevitch, C., and S. Sicouri. Clinical relevance of cardiac arrhythmias generated by afterdepolarizations: role of M-cells in the generation of U waves, triggered activity and torsade de pointes. J. Am. Coll. Cardiol. 23: 259–277, 1994. 5. Antzelevitch, C., S. Sicouri, S. H. Litovsky, A. Lukas, S. C. Krishman, J. M. Di Diego, G. A. Gintant, and D. W. Liu.


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