Repolarizing K+ Currents ITO1 and IKs Are Larger in Right Than Left Canine Ventricular Midmyocardium
Background—The ventricular action potential exhibits regional heterogeneity in configuration and duration (APD). Across the left ventricular (LV) free wall, this is explained by differences in repolarizing K+ currents. However, the ionic basis of electrical nonuniformity in the right ventricle (RV) versus the LV is poorly investigated. We examined transient outward (ITO1), delayed (IKs and IKr), and inward rectifier K+ currents (IK1) in relation to action potential characteristics of RV and LV midmyocardial (M) cells of the same adult canine hearts.
Methods and Results—Single RV and LV M cells were used for microelectrode recordings and whole-cell voltage clamping. Action potentials showed deeper notches, shorter APDs at 50% and 95% of repolarization, and less prolongation on slowing of the pacing rate in RV than LV. ITO1 density was significantly larger in RV than LV, whereas steady-state inactivation and rate of recovery were similar. IKs tail currents, measured at −25 mV and insensitive to almokalant (2 μmol/L), were considerably larger in RV than LV. IKr, measured as almokalant-sensitive tail currents at −50 mV, and IK1 were not different in the 2 ventricles.
Conclusions—Differences in K+ currents may well explain the interventricular heterogeneity of action potentials in M layers of the canine heart. These results contribute to a further phenotyping of the ventricular action potential under physiological conditions.
Regional heterogeneity of the action potential configuration and duration (APD) characterizes the ventricular myocardium in large mammals, including humans.1 2 3 A prominent notch shapes the typical spike-and-dome action potential of the epicardium and midmyocardium (M layer) but is absent in the endocardium. A relatively large transient outward current, containing a 4-aminopyridine–sensitive component (ITO1) and Ca2+-activated Cl− current, is mainly responsible for this notch. Another in vitro electrophysiological distinction is the longer APD of midmyocardium and its pronounced increase in response to slow pacing rates and class Ia and class III agents.1 4 5 These repolarization characteristics have been explained on the basis of a lesser contribution of the slowly activating component (IKs) of the delayed rectifier K+ current in M cells,4 6 whereas the rapidly activating component (IKr) and the inward rectifier current (IK1) appear similar in the 3 transmural layers.7
Only limited information is available on action potential and ionic differences in right ventricular (RV) versus left ventricular (LV) comparisons. A larger ITO1 in RV versus LV epicardial cells has been correlated with a larger notch in the former cell type.8 Because an interventricular comparison of K+ currents in M cells is lacking, we examined action potentials and the K+ currents ITO1, IKs, IKr, and IK1 in RV and LV M cells of the same adult canine hearts.
Sixteen mongrel dogs of either sex (26±1 kg) were anesthetized and received perioperative care as described previously.9 Thoracotomy was performed, and hearts (weight, 225±12 g) were quickly excised. RV and LV M cells were obtained by simultaneous cannulation of the left anterior descending and right coronary arteries.10 After ≈30 minutes of collagenase perfusion, the epicardial surface layer was removed from both wedges until a depth of ≥3 mm was reached,4 7 and softened tissue samples were removed by pipette from the M layer underneath while contamination with the endocardium was avoided. Samples were gently agitated, filtered, and washed. Isolated myocytes were stored at room temperature in standard buffer solution.
The setup was built around an inverted microscope.10 Microelectrodes (standard glass) had resistances of 30 to 60 MΩ when filled with 3.0 mol/L KCl. Intracellular pacing was done at various cycle lengths (CLs). For the recording of ionic currents, we used the whole-cell variant of the patch-clamp technique. Patch pipettes (borosilicate glass) had resistances of 1.0 to 3.0 MΩ when filled with internal solution. Experiments were performed at 37°C. Cell capacitance, measured by hyperpolarizing steps from −60 mV, was similar in RV (n=27) and LV (n=25) M cells, being 226±12 and 226±11 pF, respectively (P=NS). L-type Ca2+ current was blocked with nifedipine (5 μmol/L). Na+ current was inactivated by 10-ms prepulses to −45 mV. The voltage-clamp protocols are illustrated in Figures 1⇓ and 2⇓. ITO1 amplitudes were measured as peak amplitudes minus steady-state values at the end of the test pulses (Vtest). For IKr, we measured the tail currents on repolarization to −50 mV sensitive to almokalant (2 μmol/L; a specific IKr blocker).11 For IKs, we measured the almokalant-insensitive tail currents on repolarization to −25 mV. For IK1, we measured steady-state values at the end of Vtest.
The standard-buffer solution used for the experiments was composed of (in mmol/L) NaCl 145, KCl 4.0, CaCl2 1.8, MgCl2 1.0, NaH2PO4 1.0, glucose 11, HEPES 10, pH 7.4 with NaOH at 37°C. The patch-pipette solution contained (in mmol/L) potassium aspartate 125, KCl 20, MgCl2 1.0, MgATP 5, HEPES 5, EGTA 10, pH 7.2 with KOH.
Data are expressed as mean±SEM. Intergroup comparisons were made with the Student’s t test for unpaired and paired data groups, after testing for the normality of distribution. Differences were considered significant if P<0.05.
Action Potential Characteristics
Typical examples of RV and LV M action potentials are shown in Figure 1⇑. Quantitative data are given in the Table⇓. RV M cells had a more pronounced spike-and-dome configuration than LV M cells at fast and slow pacing rates.1 Both the action potential upstroke and plateau (phase 0 and phase 2 amplitude) were larger in LV M cells, but only at fast rates. On average, action potentials were of shorter duration in RV than LV, and they displayed less prolongation (absolutely as well as relatively) on increasing the CL from 500 to 4000 ms.
Properties of ITO1
ITO1 activated at Vtest ≥ −20 mV in both ventricles, but amplitudes were significantly larger in RV than LV at all Vtest (Figures 1⇑ and 2A⇑). 4-Aminopyridine (5 mmol/L) nearly completely suppressed ITO1 in both cell types. Inactivation during the Vtest was best fitted with a single exponential function yielding similar time constants for RV and LV. The voltage dependence of ITO1 steady-state inactivation (Figure 2B⇑) was well described by a Boltzmann fit with half points (V0.5) of −52±0.6 and −50±0.5 mV and slope factors of 6.8±0.6 and 4.5±0.5 mV in RV and LV, respectively (P=NS). Time-dependent recovery from inactivation was not different between the ventricles.
Properties of IKs and IKr
IKs tail currents were evaluated on repolarization to −25 mV with IKr blocked by almokalant. Examples of current traces are shown in Figure 1⇑. Pooled data are given in Figure 2C⇑. There was no saturation of tail-current amplitudes. Voltage dependence of IKs activation was similar for both cell types, but density was significantly larger in RV (0.72±0.12 pA/pF) than in LV (0.38±0.13 pA/pF) (P<0.05; depolarization to 50 mV). This difference persisted after increasing IKs in K+-free solution (0 [K+]O): 0.98±0.21 pA/pF in RV versus 0.58±0.17 pA/pF in LV. Deactivation proved similar in RV and LV myocytes. Tail currents in 0 [K+]O were best fitted by biexponential functions on repolarization to −10 to −40 mV and by monoexponential functions on more negative repolarizations (−50 to −80 mV). At −20 mV, time constants of the fast and slow components were 228±25 and 1105±199 ms in RV (n=7) and 278±35 and 1486±269 ms in LV (n=6), respectively; at −60 mV, monoexponential time constants were 99±16 in RV and 94±11 in LV (P=NS for all).
IKr was quantified as the almokalant-sensitive tail-current portion measured by digital subtraction at −50 mV in 4.0 mmol/L [K+]O (Figure 2D⇑). Activation showed saturation at conditioning voltages >20 mV. Boltzmann fits to the data revealed V0.5 of 2.9±1.0 and 4.3±2.5 mV in RV and LV, respectively, while corresponding slope factors were 6.2±2.1 and 5.3±0.8 mV (P=NS). IKr density was not different between RV and LV M cells. Voltage dependence and time course of IKr deactivation were also not different.
Properties of IK1
Whole-cell recordings of IK1 are shown in Figure 1⇑. IK1 rapidly activated and showed inactivation at the more negative voltages. In all cases, this current was fully inhibited in 0 [K+]O. There were no differences in the magnitude of IK1 (initial minimal values as well as steady-state levels) between RV and LV throughout the voltage range tested (Figure 2E⇑).
For interventricular comparisons of action potentials and K+ currents, we isolated myocytes from the deep subepicardial layers of the RV and LV free wall of the same canine hearts. In both ventricles, these myocytes have been designated M cells on the basis of distinctive electrophysiological characteristics.1 3 4 5 6 Our results show that action potentials have a deeper notch, a shorter duration, and less prolongation on slowing of the pacing rate in RV than in LV M cells. A longer APD in the LV versus RV has already been recorded in dogs, both in vitro1 8 and in vivo (in dogs with complete atrioventricular block).9 In 6 dogs with sinus rhythm (CL, 507±32 ms), we found endocardial monophasic APDs to be longer in LV than RV in all animals (219±6 versus 203±6 ms; P<0.05).12 Taken together, these data indicate that a larger LV than RV APD exists at normal heart rates and during bradycardia.
The presence of IKr and IKs was confirmed in M cells from the LV and was also demonstrated in RV M cells. Densities of IKr were similar in both ventricles. IKs density however, was significantly larger in RV, and this difference could explain, at least in part, why APD50 and APD95 were longer and why the APD/pacing CL relationship was steeper in LV than in RV M cells. Heterogeneity of IKs across the transmural LV wall has been linked to dispersion of repolarization and the danger of torsade de pointes.4 6 Our results on IKs (and ITO1) suggest that arrhythmogenic electromotive gradients could also arise at the septal junction of the RV and LV.
In human ventricular myocytes, the presence of IKr and IKs has also been demonstrated.13 Interestingly, Li et al13 made their observations in apparently undiseased RV myocytes of patients with left-sided heart failure. The finding of substantial amplitudes of IKr and IKs, as well as the sensitivity of both components to their blockers (E-4031 and indapamide), may underscore the importance of these currents for human ventricular repolarization, as expected from the clinical response to class Ia and class III agents in patients and from molecular studies on K+ channels in human myocardial tissue.
Our finding of a large ITO1 in RV M cells is in keeping with the prominent spike-and-dome morphology of the action potentials. Yan and Antzelevitch14 presented evidence that the distribution of ITO1 across the canine ventricular wall is causally linked to the J wave of the ECG. The joint results of this and another study8 indicate that a large ITO1-mediated notch can be found throughout most of the RV mass, which suggests that the contribution of the RV to the formation of the J wave on the ECG may be larger than previously assumed. Furthermore, this may have important consequences for our understanding of the Brugada syndrome. ST-segment elevation in the right precordial ECG leads of patients suffering from this disorder has been linked to the concept of “all-or-none repolarization” in the RV epicardium.15 If our data are applicable to patients, then the substrate predisposed to all-or-none repolarization may cover most of the RV transmural wall.
- Received September 21, 1998.
- Revision received November 4, 1998.
- Accepted November 12, 1998.
- Copyright © 1999 by American Heart Association
Antzelevitch C, Sicouri S, Lukas A, Nesterenko VV, Liu DW, Di Diego JM. Regional differences in the electrophysiology of ventricular cells: physiological and clinical implications. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: WB Saunders Co; 1995:228–245.
Li GR, Feng J, Yue L, Carrier M. Transmural heterogeneity of action potentials and Ito1 in myocytes isolated from the human right ventricle. Am J Physiol. 1998;275:H369–H377.
Liu DW, Antzelevitch C. Characteristics of the delayed rectifier current (IKr and IKs) in canine ventricular epicardial, midmyocardial, and endocardial myocytes: a weaker IKs contributes to the longer action potential of the M cell. Circ Res. 1995;76:351–365.
Anyukhovsky EP, Sosunov EA, Rosen MR. Regional differences in electrophysiological properties of epicardium, midmyocardium, and endocardium: in vitro and in vivo correlations. Circulation. 1996;94:1981–1988.
Gintant GA. Regional differences in IK density in canine left ventricle: role of IK,s in electrical heterogeneity. Am J Physiol. 1995;268:H604–H613.
Liu DW, Gintant GA, Antzelevitch C. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ Res. 1993;72:671–687.
Di Diego JM, Sun Z-Q, Antzelevitch C. Ito and action potential notch are smaller in left vs right canine ventricular epicardium. Am J Physiol. 1996;271:H548–H561.
Vos MA, de Groot SHM, Verduyn SC, van der Zande J, Leunissen HDM, Cleutjens JPM, van Bilsen M, Daemen MJAP, Schreuder JJ, Allessie MA, Wellens HJJ. Enhanced susceptibility for acquired torsade de pointes arrhythmias in the dog with chronic, complete AV block is related to cardiac hypertrophy and electrical remodeling. Circulation. 1998;98:1125–1135.
Volders PGA, Sipido KR, Vos MA, Kulcsár A, Verduyn SC, Wellens HJJ. Cellular basis of biventricular hypertrophy and arrhythmogenesis in dogs with chronic complete atrioventricular block and acquired torsade de pointes. Circulation. 1998;98:1136–1147.
Carmeliet E. Use-dependent block and use-dependent unblock of the delayed rectifier K+ current by almokalant in rabbit ventricular myocytes. Circ Res. 1993;73:857–868.
de Groot SHMA. Triggered Ventricular Arrhythmias in the Hypertrophied Heart: The Role of Electrophysiological and Functional Adaptations [doctoral thesis]. Maastricht, Netherlands: Maastricht University; 1998.
Li GR, Feng J, Yue L, Carrier M, Nattel S. Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. Circ Res. 1996;78:689–696.
Yan GX, Antzelevitch C. Cellular basis for the electrocardiographic J wave. Circulation. 1996;93:372–379.
Antzelevitch C. The Brugada syndrome. J Cardiovasc Electrophysiol. 1998;9:513–516.To study the ionic basis of repolarization differences between right and left ventricle, we measured transient outward (ITO1), delayed (IKs, IKr), and inward rectifier K+ currents (IK1) in relation to action potential characteristics of right and left ventricular midmyocardial cells of the same adult canine hearts. Action potentials had deeper notches and shorter durations in right ventricle than in left ventricle. ITO1 and IKs densities were significantly larger in right than in left ventricles, whereas IKr and IK1 were similar. Thus, differences in K+ currents may well contribute to the interventricular heterogeneity of repolarization in midmyocardial layers of the canine heart.