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Am J Physiol Heart Circ Physiol 292: H3043-H3051, 2007. First published February 16, 2007; doi:10.1152/ajpheart.01312.2006
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Heterogeneous ventricular chamber response to hypokalemia and inward rectifier potassium channel blockade underlies bifurcated T wave in guinea pig

Steven Poelzing and Rengasayee Veeraraghavan

Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah

Submitted 30 November 2006 ; accepted in final form 12 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It was previously demonstrated that transmural electrophysiological heterogeneities can inscribe the ECG T wave. However, the bifurcated T wave caused by loss of inward rectifier potassium current (IK1) function is not fully explained by transmural heterogeneities. Since right ventricular (RV) guinea pig myocytes have significantly lower IK1 than left ventricular (LV) myocytes, we hypothesized that the complex ECG can be inscribed by heterogeneous chamber-specific responses to hypokalemia and partial IK1 blockade. Ratiometric optical action potentials were recorded from the epicardial surface of the RV and LV. BaCl2 (10 µmol/l) was perfused to partially block IK1 in isolated guinea pig whole heart preparations. BaCl2 or hypokalemia alone significantly increased RV basal (RVB) action potential duration (APD) by ~30% above control compared with LV apical (LVA) APD (14%, P < 0.05). In the presence of BaCl2, 2 mmol/l extracellular potassium (hypokalemia) further increased RVB APD to a greater extent (31%) than LVA APD (19%, P < 0.05) compared with BaCl2 perfusion alone. Maximal dispersion between RVB and LVA APD increased by 105% (P < 0.05), and the QT interval prolonged by 55% (P < 0.05) during hypokalemia and BaCl2. Hypokalemia and BaCl2 produced an ECG with a double repolarization wave. The first wave (QT1) corresponded to selective depression of apical LV plateau potentials, while the second wave (QT2) corresponded to the latest repolarizing RVB myocytes. These data suggest that final repolarization is more sensitive to extracellular potassium changes in regions with reduced IK1, particularly when IK1 availability is reduced. Furthermore, underlying IK1 heterogeneities can potentially contribute to the complex ECG during IK1 loss of function and hypokalemia.

electrophysiology; waves; electrocardiography; interventricular heterogeneities; inward rectifier potassium current

IT IS WELL ESTABLISHED THAT electrophysiological heterogeneities inscribe the distinct ECG morphology. The T wave, representing final repolarization, has been linked to dispersion of repolarization across the ventricular wall (40). However, the preferential appearance of bifurcated T waves in leads II and V3 during hypokalemia (39) suggests that the predominant heterogeneity during hypokalemia may be regional rather than transmural.

Interestingly, hypokalemia has dichotomous effects on repolarizing potassium currents. For example, the inward rectifier potassium current (IK1) has a paradoxical relationship with extracellular K+ concentration ([K+]o) where IK1 peak current density is reduced during hypokalemia (28). IK1 plays an important role in ventricular action potentials by modulating resting membrane potential and final repolarization. In humans, IK1 reduction, as occurs in Andersen-Tawil syndrome type 1 (ATS1), is associated with prominent secondary repolarization ECG waves (U waves) particularly during hypokalemia (41, 42). While the functional IK1 protein distribution in humans remains unknown, IK1 heterogeneities have been demonstrated previously in a variety of other animal models (8, 37). However, it is unclear what role IK1 heterogeneities have in the inscription of the ECG, particularly under conditions in which IK1 is partially blocked or altered by lowering extracellular potassium.

Previous studies of partial IK1 blockade have been unable to recapitulate a bifurcated T wave with or without hypokalemia (29, 33). Interestingly, these models of partial IK1 blockade only explored the contribution of left ventricular (LV) transmural heterogeneities to the ECG's inscription. Since right ventricular (RV) guinea pig myocytes have significantly lower IK1 than LV myocytes (37), we hypothesized that the complex ECG can be inscribed by heterogeneous chamber-specific responses to hypokalemia and partial IK1 blockade.

In this study we demonstrate that the response to hypokalemia and partial IK1 blockade is heterogeneous between the RV and the LV in guinea pig. In general, action potential duration (APD) of myocytes with reduced IK1 (RV) prolongs significantly more than those with more IK1 (LV) during hypokalemia and partial IK1 blockade. This ventricular chamber-specific heterogeneity manifests as a complex ECG repolarization morphology arising from differences in action potential trajectories. Specifically, the first wave after depolarization, labeled T1 in accordance with suggested nomenclature (35), is inscribed by plateau differences in APD, and the final repolarization wave T2 is inscribed by final repolarization of cells in the base of the RV. Interestingly, the increased dispersion of repolarization between ventricles was insufficient for arrhythmia induction via programmed stimulation. However, spontaneous and rapid pacing-induced arrhythmias were observed during hypokalemia and partial IK1 blockade.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental preparation. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, Revised 1996) and has been approved by the Institutional Animal Care and Use Committee of the University of Utah (protocol no. 05-07002). Retired breeder male guinea pigs were anesthetized [30 mg/kg pentobarbital sodium (Nembutal) ip; n = 19], and their hearts were rapidly excised and perfused as Langendorff preparations (perfusion pressure 55 mmHg) with oxygenated (100% O2) Tyrode solution at 36.5°C containing (mmol/l) 2 CaCl2, 140 NaCl, 4.5 KCl, 10 dextrose, 1 MgCl2, and 10 HEPES (pH 7.40). Preparations were completely immersed in temperature-controlled (36 ± 1°C) perfusate to prevent the formation of regional temperature gradients. The right and left atria were excised to avoid competitive stimulation. Hearts were stained with the voltage-sensitive dye di-4-ANEPPS (15 µmol/l) by direct coronary perfusion for 10 min.

Ratiometric optical mapping system. To reduce motion artifacts and more accurately reconstruct transmembrane potentials, we developed a ratiometric optical action potential mapping system similar to that of Himel and Knisley (13). Specifically, we used two SciMedia MiCam02 HS charge-coupled device (CCD) cameras (SciMedia, Irvine, CA) in a tandem lens configuration capable of resolving membrane potential changes with 1-ms temporal resolution from 90 x 60 sites simultaneously sampled with a 14-bit analog-to-digital converter. After staining with the voltage-sensitive dye di-4-ANEPPS, the preparation was excited by two 60-LED light sources (RL5-A9018, Superbrightleds, St. Louis, MO) powered by a custom-built 15-V DC power supply (3 0 mA per LED). Each light source was fitted with 510 ± 5-nm filters (Chroma, Rockingham, VT) and a 50-mm aspheric lens (Edmund Optics, Barrington, NJ). Fluoresced light passed through a 150-mm achromatic lens (BK7/Flint, Ealing, Rocklin, CA) and was incident on a 565DXR dichroic mirror (Chroma) set at a 45° angle to the recording surface. Transmitted light passed through a 50-mm aspheric B270 crown glass lens (Edmund Optics), a 35-mm planoconvex BK7 lens (Edmund Optics), and a 610-nm LP filter (Newport, Irvine, CA) before it was incident on the CCD array. Reflected light passed through a 50-mm aspheric B270 crown glass (Edmund Optics), a 35-mm planoconvex BK7 lens (Edmund Optics), and a 540 ± 10-nm filter (Chroma) where it was incident on the second CCD array. CCD arrays were optically aligned at fixed and equal optical path lengths. The interpixel resolution was 0.184 mm in the x-direction (90 pixels) and 0.199 mm in the y-direction (60 pixels). The relative change in voltage (Vm) was defined as Vm = {Delta}F610/{Delta}F540, where {Delta}F represents the change in fluorescence for a particular wavelength.

Optical action potential measurements. Motion was further reduced by perfusion of 7.5 mmol/l 2,3-butanedione monoxime. Hearts were stimulated with a unipolar silver wire placed on the basal epicardial septum at 1.5 times the stimulation threshold with basic cycle lengths (BCL) of 500 Hz down to loss of 1:1 capture. Activation time was defined as the time of the maximum first derivative of the action potential as described previously (11). Repolarization was defined as the time at which the amplitude of the voltage transient reached 95% repolarization from peak voltage amplitude. APD was the time difference between activation and repolarization.

All recordings were divided into eight regions from the LV and RV corresponding to their anatomic location (anterior: RV base, RV apex, LV base, LV apex; posterior: RV base, RV apex, LV base, LV apex). Each region consisted of a minimum of 400 optical sites. Regional APD was quantified as the average regional APD. Anterior and posterior measurements were made sequentially by turning the heart in the imaging chamber in order to record from appropriate sites.

ECG measurements. The bath ECG was obtained with a silver chloride anode located ~2 cm from the midwall of the RV and a cathode located ~2 cm from the midwall of the LV. QT1 and QT2 were calculated as the time difference between initial depolarization following the stimulus and the end of the T1 or T2 wave. The QT1 and subsequent QT2 intervals were plotted, and regression analysis was performed for each QT correction equation obtained from Malik et al. (22). The hyperbolic QT correction formula [QTc = QT + (1/BCL – 1)] demonstrated the best fit for all measurements (slope < 0.1, P < 0.01) and was therefore used to compare the QT/QU relationship over all BCL.

Pharmacological intervention. Since BaCl2 is a relatively specific IK1 blocker at micromolar doses (30), we varied extracellular BaCl2 from 10 to 100 µmol/l (n = 3). A final concentration of 10 µmol/l BaCl2 (n = 6) was chosen to determine the role of [K+]o on action potential trajectories during IK1 blockade. This concentration is similar to that used in a canine model of IK1 blockade (33) and a guinea pig model of ventricular fibrillation (37). We varied [K+]o in the presence and absence of BaCl2 (n = 6 additional experiments). All optical recordings were made after 15 min of BaCl2 and/or 2 (hypokalemic), 4.5 (normokalemic), or 6 (hyperkalemic) mmol/l [K+]o perfusion. All interventions were compared with control conditions (n = 4).

Arrhythmia induction. To assess changes in susceptibility to arrhythmias during all experimental conditions, programmed electrical stimulation was performed according to an identical protocol on all preparations. After a 20-beat drive train delivered to the anterior epicardial surface of either the RV or LV (BCL of 400 ms), an epicardial premature stimulus (S2) was delivered through the same drive train electrode at a S1-S2 coupling interval of 300 ms. The S1-S2 interval was sequentially shortened by 10-ms decrements until refractoriness was reached or an arrhythmia was induced. Subsequently, a 20-beat drive train was delivered at a BCL 20 ms longer than the shortest pacing rate capable of 1:1 capture in order to observe arrhythmia susceptibility to rapid pacing.

Statistical analysis. Statistical analysis of the data was performed with a two-tailed Student's t-test for paired and unpaired data or a single-factor ANOVA. A P < 0.05 was considered statistically significant. All values are reported as means ± SE unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Regional APD heterogeneity. For all control experiments anterior RV basal (RVB) APD was significantly greater than that in all other regions of the heart (Table 1). Importantly, anterior RVB and anterior RV apical APD were significantly greater than the corresponding posterior regions (posterior RV base and posterior RV apex). In the LV, only the anterior LV basal (LVB) APD was significantly greater than the posterior LVB APD. Extracellular BaCl2 was varied from 10 to 100 µmol/l in order to determine the regional heterogeneity of APD during IK1 blockade. Representative action potentials in Fig. 1A demonstrate the effect of 30 and 100 µmol/l BaCl2. Specifically, APD for all cell types significantly prolonged with increased BaCl2 (BCL = 350 ms). Furthermore, the QT interval obtained from the bath ECG prolonged with increased BaCl2 as demonstrated in Fig. 1A. The graph in Fig. 1B demonstrates that the mean APD for all experiments increases as the concentration of BaCl2 is increased. The APD maximum regional dispersion likewise increased as BaCl2 concentration increased (Fig. 1C).


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  		Table 1. Regional APD under control conditions and during perfusion of 10 µmol/l BaCl2

 

Figure 1
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  		Fig. 1. Inward rectifier potassium current (IK1) blockade increases action potential duration (APD) and APD dispersion. A: representative action potentials from the anterior surface of guinea pig ventricle. Increased doses of BaCl2 increase APD for all epicardial cell types. Increased APD is associated with increased ECG QT interval. B: mean APD for the entire anterior epicardial surface increases with increased concentration of BaCl2. C: regional APD dispersion [left ventricular apex (LVA) subtracted from right ventricular base (RVB) APD] increases with blockade of IK1. *P < 0.05 compared with 0 µmol/l BaCl2.

 
To compare these results with previous canine models of partial IK1 blockade (33) and electrophysiological changes associated with BaCl2 perfusion in guinea pig ventricles (37), a 10 µmol/l dose of BaCl2 was used for subsequent experiments. The regional distribution pattern of APD remained similar during BaCl2 perfusion (Table 1). However, APD significantly prolonged for all regions (P < 0.05). Additionally, maximal APD dispersion occurred between the anterior RVB and the anterior LV apex (LVA) (Table 1) and was increased compared with control by 54%.

Hypokalemia and IK1 blockade increase interventricular heterogeneity. Since extracellular potassium fluctuations are often associated with ventricular ectopy, extracellular potassium was varied to 6 and 2 mmol/l [K+]o in the presence of 10 µmol/l BaCl2. Representative action potentials in Fig. 2A demonstrate the change in APD from corresponding sites in Fig. 2B. For all regional cell types, BaCl2 (Fig. 2A, red traces) increased APD above control (Fig. 2A, black traces). During BaCl2 perfusion, APD varied inversely with [K+]o. Specifically, 6 mmol/l [K+]o + BaCl2 (Fig. 2A, blue traces) decreased APD for all cell types compared with 4.5 mmol/l [K+]o + BaCl2. On the other hand, 2 mmol/l [K+]o + BaCl2 (Fig. 2A, green traces) increased APD for all regions compared with control and 4.5 mmol/l [K+]o + BaCl2.


Figure 2
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  		Fig. 2. Hypokalemia and 10 µmol/l BaCl2 increase regional APD heterogeneity. A: for all regions of the heart, addition of 10 µmol/l BaCl2 (red traces) increases APD above control (black traces). In the presence of BaCl2, lowering extracellular potassium concentration ([K+]o) to 2 mmol/l (green traces) significantly increases APD for all regions above all other experimental conditions. Raising [K+]o to 6 mmol/l (blue traces) in the presence of 10 µmol/l BaCl2 decreases APD from 4.5 mmol/l [K+]o for all regions. LVB, left ventricular base; RVA, right ventricular apex. B: control APD distribution demonstrates a gradient from RVB to LVA (top left). Addition of BaCl2 causes all regional APD to increase and accentuates the RVB-to-LVA gradient (top right). BaCl2 and hyperkalemia increase all regional APD but attenuate regional gradients (bottom right). Hypokalemia and BaCl2 increase all regional APD greater than any other experimental condition and produce the largest APD gradients in this study (bottom left).

 
The representative APD maps in Fig. 2B demonstrate the spatial distribution of APD on the anterior surface of the heart during experimental conditions. Maps were scaled such that the APD range for all experimental conditions in Fig. 2B is constant (45 ms). The anterior RVB manifests the longest APD compared with all other regions of the heart, as mentioned above. Additionally, BaCl2 significantly increased APD for all regions (Fig. 2B, top right) as demonstrated by an increase in sites with the shortest APD from 150 ms under control conditions to 170 ms. BaCl2 increased APD dispersion from the control value of 23 ms to 35 ms. Hyperkalemia (6 mmol/l [K+]o) and BaCl2 decreased regional dispersion back to 22 ms (Fig. 2B, bottom right) while maintaining a significantly greater average APD over control. Importantly, 2 mmol/l [K+]o + BaCl2 further increased APD for all cell types, consistent with the paradoxical dependence of IK1 (12) and the rapid component of delayed rectifier potassium current (IKr) (27) on [K+]o. Additionally, APD dispersion increased to 45 ms (Fig. 2B, bottom left).

Maximum APD dispersion occurred between anterior RVB and anterior LVA for all experiments and conditions. In the results described below, RVB refers to all action potentials recorded from the anterior RVB, and LVA refers to all action potentials recorded from the anterior LVA.

Summary data in Fig. 3A demonstrate that under baseline conditions RVB APD is significantly greater than LVA APD for all [K+]o (P < 0.05). BaCl2 significantly increased APD in both regions for all [K+]o (P < 0.05). However, RVB APD was significantly greater than LVA APD only during perfusion of 2 and 4.5 mmol/l [K+]o. Hyperkalemia attenuated regional APD heterogeneities in the presence of BaCl2.


Figure 3
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  		Fig. 3. Summary of regional APD during varying [K+]o and BaCl2. A: average APD from the anterior base of right and left ventricle (RVB and LVA) during baseline conditions and perfusion of 10 µmol/l BaCl2. APD for all cell types demonstrates an inverse relationship to [K+]o. This inverse dependence is increased during perfusion of BaCl2. RVB APD is greater than LVA APD in control and BaCl2-perfused experiments ({dagger}P < 0.05) except during 6 mmol/l [K+]o + BaCl2. BaCl2 increases all APD above baseline for all cell types (*P < 0.05 compared with corresponding baseline values). B: difference of regional APD for all experiments during baseline conditions demonstrates that heterogeneity is significantly increased only during perfusion of 2 mmol/l [K+]o compared with 4.5 mmol/l [K+]o ({dagger}P < 0.05). This relationship is also present during perfusion of BaCl2. Addition of BaCl2 significantly increases all regional APD differences (*P < 0.01) for 2 and 4.5 mmol/l [K+]o.

 
Regional APD dispersion is summarized in Fig. 3B. Hypokalemia in the absence of BaCl2 significantly increased APD dispersion above 4.5 mmol/l [K+]o by ~50%. However, hyperkalemia alone has no observable affect on APD dispersion. BaCl2 increased APD dispersion under conditions of 2 and 4.5 mmol/l [K+]o. Specifically, hypokalemia increased APD dispersion by ~105% during BaCl2 perfusion, while 4.5 mmol/l [K+]o + BaCl2 increased APD to a similar extent as hypokalemia alone (~30%, Fig. 3B). Again, hyperkalemia did not significantly change APD dispersion in the presence of 10 µmol/l BaCl2.

ECG manifestations during hypokalemia and IK1 blockade. The measurement of the QT interval obtained from the bath ECG during control conditions is illustrated in Fig. 4A. Under control conditions, pacing from the anterior basal septum produces a bath ECG pattern with a well-defined QRS and T-wave morphology (Fig. 4A). Hypokalemia (Fig. 4B) prolongs the QT interval and causes ST-segment depression. The addition of BaCl2 under normokalemic conditions increases the QT interval but otherwise does not significantly change the morphology of the bath ECG (Fig. 4C). Hypokalemia and BaCl2 are associated with ST-segment depression and the appearance of an additional repolarizing wave labeled T2 in Fig. 4D. However, the end of T2 in this example is obscured by the pacing artifact for the subsequent beat. Prolonging the BCL to 400 (Fig. 4E) and 500 (Fig. 4F) ms reveals final repolarization of T2.


Figure 4
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  		Fig. 4. Bath ECG morphology. A: normal Tyrode perfusion is associated with a well-defined QRST morphology. B: hypokalemia produces modest ST-segment depression and QT prolongation. C: BaCl2 + 4.5 mmol/l [K+]o increases the QT interval but does not affect the ST segment. D: decreasing extracellular potassium in the presence of BaCl2 is associated with a double repolarization wave. The first wave with ST segment depression is labeled as the T1 wave since it is the first wave following the QRS, and the second deflection as the T2 since it follows an isoelectric point on the ECG. E and F: prolonging the basic cycle length (BCL) to 400 (E) and 500 (F) ms demonstrates that the portion after T2 reaches an isoelectric window preceding the following beat. Locations for the measurement of the QT1 and QT2 intervals measured in Fig. 5 are demonstrated. G: recording of a spontaneously initiated polymorphic ventricular tachycardia during hypokalemia and BaCl2 perfusion. H: representative nonsustained arrhythmia induced by rapid pacing during hypokalemia and BaCl2 perfusion.

 
QT1c for all experiments and pacing rates from 500- to 300-ms BCL are summarized in Fig. 5A. Normokalemia with BaCl2 significantly increased QT1c from 185 ± 4 ms under control conditions to 225 ± 2 ms (P < 0.001). Hypokalemia with BaCl2 did not significantly change QT1c (184 ± 5 ms) compared with control. The QT2c interval under conditions of hypokalemia with BaCl2 significantly increased above control and normokalemia with BaCl2 perfusion. The latest repolarization times (top 95th percentile) in the RVB (285 ± 13 ms) closely corresponded to QT2c (280 ± 12 ms, Fig. 5B). However, the earliest repolarization times (lowest 5th percentile) in the LVA (251 ± 13) were significantly longer than QT1c (P < 0.05).


Figure 5
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  		Fig. 5. QT1c does not follow typical patterns associated with blocking repolarizing currents. A: BaCl2 perfusion significantly increases QT1c (*P < 0.001) as expected above control. However, 2 mmol/l [K+]o + BaCl2 does not significantly change QT1c compared with QTc. On the other hand, the QT2c (black bar) is significantly greater than control and 4.5 mmol/l [K+]o + BaCl2. B: the longest repolarization times in the RVB (top 95%) during 2 mmol/l [K+]o and BaCl2 perfusions are significantly greater than the shortest repolarization times in the LVA (lowest 5%) (*P < 0.05). The shortest repolarization times in the LVA are significantly greater than QT1c during 2 mmol/l [K+]o and BaCl2 perfusion ({dagger}P < 0.05). C: action potentials from RVB and LVA are superimposed to demonstrate differences in trajectory and APD (top). Digital subtraction of the top traces yields an action potential trajectory gradient trace (middle) that demonstrates 2 repolarization waves corresponding to the regional potential differences growing greater, coming together, and then diverging again during phases 2 and 3 of the action potential. The bath ECG (bottom) recapitulates the double repolarization wave observed in the middle trace.

 
Representative action potentials from RVB and LVA during 2 mmol/l [K+]o + BaCl2 perfusion are superimposed in Fig. 5C. The first phase of repolarization in LVA sites is significantly faster than that in RVB sites. Digital subtraction of these two traces reveals a double repolarization wave difference. This double wave corresponds well with the double repolarization wave observed in the bath ECG under these conditions. Therefore, these data suggest that T1, under conditions of 2 mmol/l [K+]o + BaCl2 perfusion, represents interventricular plateau heterogeneities and T2 represents final repolarization.

Action potential trajectory changes independent of IKr. Varying extracellular potassium has dichotomous effects on repolarizing currents. Specifically, IKr has a paradoxical relationship with [K+]o, in which low [K+]o decreases IKr. On the other hand, the slow component of the delayed rectifier potassium current (IKs) has an ohmic relationship with [K+]o, in which low [K+]o increases IKs (27). Since IKr has a paradoxical relationship to [K+]o, our results with hypokalemia and BaCl2 may also unveil heterogeneities due to partial IKr block and produce an complex ECG. Therefore, it was important to determine the effects of IKr blockade during hypokalemia on the ECG.

Regional APD differences during hypokalemia, represented as APD dispersion in Fig. 6A, significantly increased with the addition of BaCl2. The underlying mechanism of increased APD dispersion is a result of hypokalemia and BaCl2 significantly prolonging RVB APD to a greater extent than LVA APD (Fig. 3A; P < 0.05). Terfenadine, a potent IKr blocker, and 2 mmol/l [K+]o also significantly increased regional APD dispersion above 2 mmol/l [K+]o alone and 2 mmol/l [K+]o + BaCl2 (Fig. 6B). The largest dispersion in this experiment likewise occurred between RVB and LVA. Representative action potentials from RVB and LVA are superimposed in Fig. 6B. The trajectories of the two action potentials are similar. Action potentials in both regions appear more triangulated, and terfenadine and hypokalemia increase RVB APD to a greater extent than LVA APD. The action potential difference trace and bath ECG (Fig. 6B) demonstrate a single repolarization wave.


Figure 6
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  		Fig. 6. APD dispersion increases but morphology does not change when blocking the rapid component of the delayed rectifier potassium current (IKr) during hypokalemia. A: APD dispersion is significantly increased during 10 µmol/l BaCl2 (IK1 blocker) and terfenadine (IKr blocker) perfusion (*P < 0.001); 2 µmol/l terfenadine significantly increases APD dispersion above BaCl2. B: action potentials from RVB and LVA are superimposed to demonstrate differences in morphology and APD (top). Digital subtraction of the top traces yields a {triangledown} action potential morphology (middle) that demonstrates a single repolarization wave corresponding to the differences in final repolarization of the action potential. The bath ECG (bottom) recapitulates the single repolarization wave observed in the middle trace and does not display a T2 wave. This is consistent with similar action potential trajectories in the top traces except for final repolarization times.

 
Ventricular arrhythmias. Arrhythmias were neither spontaneous nor initiated with programmed electrical stimulation during perfusion of BaCl2 or hypokalemia alone. However, during hypokalemia and BaCl2, spontaneous arrhythmias occurred in three of six preparations. Representative bath ECGs in Fig. 4, G and H, demonstrate a spontaneous polymorphic arrhythmia and a rapid pacing-induced monomorphic arrhythmia, respectively. The last stimulus (S1, BCL = 150 ms) is present in Fig. 4H. All arrhythmias were self-terminating. Arrhythmias were never induced with programmed stimulation (up to 2 extra premature beats) during hypokalemia and BaCl2. Relatively rapid pacing (between 310- and 250-ms BCL) produced from three extra beats to 30 s of spontaneously terminating monomorphic ventricular tachycardia in five of six preparations with hypokalemia and BaCl2. In contrast, rapid pacing did not produce any tightly coupled spontaneous beats under control conditions.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The purpose of this study was to test the hypothesis that the complex ECG can be inscribed by heterogeneous responses to hypokalemia and partial IK1 blockade due to underlying IK1 heterogeneities. We observed the greatest differences in APD between the anterior RVB and the anterior LVA under all experimental conditions, consistent with reduced RV IK1 expression (26). These data of regional APD heterogeneities from the anterior epicardial surface of guinea pig ventricles under control conditions are consistent with previous reports (19, 38). Hypokalemia, BaCl2, or terfenadine alone increased RVB APD, which corresponded well with QT prolongation. Despite QT prolongation, these interventions did not alter the ECG morphology. However, combining hypokalemia with partial IK1 blockade produced a complex ECG with two repolarization waves. Specifically, the first repolarization wave (QT1c) was not significantly different from control, while the second ECG repolarization wave (QT2c) became prominent and was significantly longer than QTc and QT1c for all experimental conditions. In this model of hypokalemia and partial IK1 blockade, QT1 corresponded to selective depression of apical LV plateau potentials compared with other regions, while QT2 corresponded to disproportionate prolongation of RVB repolarization times compared with LVA. These data suggest that final repolarization of myocytes in regions with reduced IK1 expression (RVB myocytes in the case of the guinea pig) may be more sensitive to changes in [K+]o under conditions of reduced IK1 availability. Furthermore, the complex ECG repolarization waves may be a result of selective difference in action potential trajectory and not just final repolarization under conditions of hypokalemia and reduced IK1.

It was demonstrated previously that heterogeneous transmural action potential trajectory differences in the canine wedge preparation can inscribe a bifurcated T wave during sotalol perfusion with (40) or without (41) hypokalemia. While the electrophysiological heterogeneity was transmural rather than between chambers, the mechanism of action potential trajectory differences is consistent with the mechanism of a bifurcated T wave observed in this study. However, a bifurcated T wave was not observed in the canine wedge preparation under conditions of hypokalemia and partial IK1 blockade (33).

It is important to note that the complex T wave may have more than one underlying mechanism depending on the experimental model or disease. For example, the second component of a bifurcated T wave has been referred to as a U wave in the literature (40). U waves have been observed in patients with hypokalemia (39), catecholaminergic polymorphic ventricular tachycardia (1), ischemia (7, 10, 23), and ventricular hypertrophy and dilatation (24, 34), to name a few conditions. Additionally, U waves manifest in normal subjects (21), further adding to the controversial nature of this wave. It has been postulated that muscle movement and stretch-activated channels may contribute to delayed afterdepolarizations and subsequently the U wave (32, 35). Muscular contraction and the distribution of stretch-activated channels may also be regionally heterogeneous. Therefore, the underlying mechanism of the U wave could be a result of delayed afterdepolarizations or heterogeneous repolarization. This study is consistent with previous reports that suggest the second component of the bifurcated T wave in guinea pig during hypokalemia and IK1 blockade is a "resumption of an interrupted T wave" (40), warranting the label "bifurcated T wave" instead of "U wave." Therefore, a broader investigation of the entire ventricular myocardium may be necessary to determine the underlying mechanisms of bifurcated T waves compared with U waves.

IK1 and heterogeneous chamber-specific responses. The heterogeneous ventricular chamber-specific response to hypokalemia and partial IK1 blockade is likely related to the reduced RV inward rectifier potassium channel (Kir)2.1 and Kir2.3 expression compared with LV previously demonstrated in guinea pig (26, 37). However, IK1 distribution seems to be species dependent. Specifically, there are no measurable IK1 differences in canine myocardium either transmurally (20) or between ventricles (36), while IK1 is heterogeneously distributed transmurally in feline myocardium (8). Therefore, blocking IK1 may have heterogeneous effects regionally or transmurally depending on the animal model chosen. In general, these data suggest that IK1 heterogeneities may play a larger role in electrophysiological heterogeneities and the inscription of the ECG than previously proposed.

Arrhythmia mechanisms. While total APD dispersion was significantly increased under conditions of hypokalemia, BaCl2, terfenadine, or the combination of hypokalemia and BaCl2, the magnitude of dispersion did not exceed 50 ms. Specifically, gradients of repolarization between the RV and LV were <5 ms/mm, significantly lower than the proposed value of 10 ms/mm that was previously found to be critical for the development of conduction block and reentry (2). These data are consistent with our inability to initiate arrhythmias with programmed stimulation. Furthermore, the observed interventricular and apicobasal heterogeneities may have been an insufficient substrate for arrhythmias in guinea pig, and therefore it may be that these gradients are even less sufficient to serve as a substrate for arrhythmias in the case of larger hearts since the repolarization gradients between ventricles may be less steep. A previous study of IK1 blockade in the canine wedge preparation demonstrated that LV transmural gradients of repolarization were also insufficient to induce arrhythmias, but triggered activity was increased during hypokalemia and partial IK1 blockade (33).

Hypokalemia and cycle length prolongation before arrhythmia initiation, on the other hand, are common clinical features of drug-induced torsade de pointes (5, 25, 31), where afterdepolarization activity increases and serves as the arrhythmia trigger. We observed spontaneous arrhythmias only during hypokalemia and partial IK1 blockade. However, it remains unknown whether the spontaneous arrhythmias were reentrant or focal in nature. In this study, the initiations of monomorphic arrhythmias via rapid pacing only occurred during hypokalemia and partial IK1 blockade, which is in conflict with bradycardia-induced torsade de pointes. Rapid pacing is also associated with triggered activity due to elevation of diastolic calcium levels (18), and hypokalemia alone can raise intracellular diastolic calcium levels and thereby increase triggered activity (9). While IK1 downregulation up to ~80% is sufficient to significantly increase pacemaker activity in ventricular myocytes, it remains unknown whether a much smaller degree of IK1 block can lower the threshold for triggered activity, particularly during hypokalemia when IK1 is further reduced. This study suggests that arrhythmias during hypokalemia and partial IK1 blockade may be a result of triggered activity and less likely dependent on large gradients of repolarization acting as a substrate for reentrant arrhythmias.

IK1 reduction and disease. While it is tempting to link our results to ATS1, a disease characterized by a loss of IK1 function due to mutations in KCNJ2, this interpretation is complicated by species heterogeneity and experimental protocols. Specifically, the regional distribution of IK1 in human ventricular myocardium remains unknown. Second, all measurements in this study were made during epicardial pacing. As a result, QRS and the bifurcated T-wave concordance or discordance cannot be directly compared with the complex ECG obtained in ATS1 patients during sinus rhythm. As mentioned above, the underlying mechanism for inscribing a bifurcated T wave or a U wave may vary between species and experimental protocols, further complicating comparison. Additionally, there are well-established differences in potassium currents between guinea pigs and other larger species such as humans. For example, the guinea pig ventricular myocardium does not functionally express a 4-aminopyridine-sensitive transient outward current (Ito) (4, 14–16). Finally, the heart rate for a guinea pig is significantly faster than a human's, and the mild QTc prolongation observed in ATS1 patients may not be directly comparable with QTc intervals observed in this study. However, understanding the role IK1 heterogeneities play in general in cardiac electrophysiology will yield greater insights into not only ATS1 but also diseases that result in a loss of IK1 function such as heart failure (17).

Limitations. While BaCl2 is a relatively potent IK1 blocker, barium alters calcium-mediated inactivation of the L-type calcium channel (6). However, this study used doses of BaCl2 orders of magnitude smaller (µmol/l) than those reported to significantly alter L-type calcium channel kinetics (mmol/l) (3). Like all nonspecific blockers, the potential for nonspecific electrophysiological effects may complicate interpretation. Therefore, this study should not be considered a direct model of any specific IK1 loss-of-function disease. Instead, these results highlight the importance of considering chamber-specific heterogeneities that can underlie complex ECG phenomenon.


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This study was supported by a grant from the Nora Eccles Treadwell Foundation (to S. Poelzing).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Poelzing, Nora Eccles Harrison Cardiovascular Research and Training Institute, Univ. of Utah, 95 South 2000 East, Salt Lake City, UT 84112-5000 (e-mail: poelzing{at}cvrti.utah.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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