IK1 Heterogeneity Affects Genesis and Stability of Spiral Waves in Cardiac Myocyte Monolayers
Previous studies have postulated an important role for the inwardly rectifying potassium current (IK1) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1expression achieved by lentiviral transfer to elucidate the role of IK1 in cardiac arrhythmogenesis. Kir2.1-overexpressed NRVMs showed increased IK1 density, hyperpolarized resting membrane potential, and increased action potential upstroke velocity compared with green fluorescent protein–transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed altered conduction velocity and action potential duration compared with nontransduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with an island of altered Kir2.1 expression, conduction velocity and action potential duration of the locally transduced and nontransduced regions were similar to those of the uniformly transduced and nontransduced monolayers, respectively, and functional reentrant waves could be induced. The waves were anchored to islands of Kir2.1 overexpression and remained stable but dropped in frequency and meandered away from islands of Kir2.1 suppression. In monolayers with an inverse pattern of IK1 heterogeneity, stable high frequency spiral waves were present with IK1 overexpression, whereas lower frequency, meandering spiral waves were observed with IK1 suppression. Our study provides direct evidence for the contribution of IK1 heterogeneity and level to the genesis and stability of spiral waves and highlights the potential importance of IK1 as an antiarrhythmia target.
- inwardly rectifying potassium current
- spiral waves
- ventricular tachycardia
- ventricular fibrillation
Ventricular fibrillation (VF) is the leading cause of cardiac arrest and sudden cardiac death in the industrialized world.1 Studies in the 1970s suggested that the heart could sustain electrical activity that rotated around a functional obstacle.2,3 These reentrant waves are believed to be the unitary components of fibrillation. Several other studies that focused on understanding the mechanisms of initiation and maintenance of VF concluded that the stability of spiral waves (functional form of reentrant waves) depends on the abbreviation of action potential duration, as well as the reduction of wavefront–wavetail interactions, at fibrillation frequencies.4–6 In addition, ionic heterogeneity may be a key factor in the initiation of spiral waves and their transition to the irregular spatiotemporal pattern seen in VF.7–9 Numerous studies pioneered mainly by Jalife and colleagues have indicated that IK1 plays an important role in determining cardiac excitability and arrhythmogenesis and that IK1 block has a significant effect on VF dynamics.4,5,10–13 Also, genetic mutations in Kir2.1, the molecular correlate of IK1, can cause short QT syndrome SQT314 and Andersen syndrome15 with accompanying ventricular arrhythmias. Although previous studies in transgenic mice have concluded that IK1 upregulation stabilizes high-frequency rotors (organizing center of spiral waves),11,13 Kir2.1 expression in such transgenic hearts can be heterogeneous,16 and the effects of such heterogeneity on arrhythmogenesis have not been explored. We therefore investigated the effects of altered Kir2.1 expression on spiral wave dynamics in a novel, in vitro model of cultured neonatal rat ventricular myocytes (NRVMs) in which regional variations of Kir2.1 were engineered by tissue engineering and somatic gene transfer approaches. In NRVM monolayers with distinct regions of Kir2.1 overexpression or dominant-negative suppression, the role of IK1 heterogeneity in initiating and maintaining a high-frequency spiral wave was studied. Also, the secondary effects of IK1 modulation on cardiac single-cell and tissue electrophysiological properties, such as resting membrane potential (RMP), action potential (AP) phenotype, maximum upstroke velocity (dV/dtmax), action potential duration (APD), conduction velocity (CV), and maximum capture rate were studied. The results of our study demonstrate that uniform upregulation of IK1 does not necessarily create a medium that is prone to arrhythmogenesis and suggest that CV and APD differences arising from heterogeneous overexpression of Kir2.1 contribute to the genesis and stability of reentrant spiral waves that can underlie life-threatening cardiac arrhythmias.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. In brief, freshly isolated, nontransduced NRVMs and NRVMs transduced by empty lentiviral vectors (LV-Empty) or lentiviral vectors (LVs) encoding Kir2.1 (LV-Kir2.1) or Kir2.1AAA (LV-Kir2.1AAA) were plated on 12-mm fibronectin-coated glass (for patch clamping) or 21-mm fibronectin-coated plastic (for optical mapping) coverslips as previously described.17 A modified form of a stenciling technique,18 involving polydimethylsiloxane stamps, was used to spatially localize transduced NRVMs in a coculture model with nontransduced NRVMs. Six-day-old monolayers were characterized by immunostaining for cardiac troponin (cTn)I, actin, Kir2.1, and connexin (Cx)43. The levels of Kir2.1 and Cx43 were also characterized by Western blot. The electrophysiological single-cell properties of Kir2.1 gene-modified NRVMs were characterized by standard microelectrode whole-cell patch clamp techniques. Optical mapping over a 17-mm-diameter field of view was performed on 6-day-old, 21-mm-diameter isotropic monolayers and monolayers with heterogeneous Kir2.1 expression to study the electrophysiological tissue properties. Bipolar line stimulation via platinum electrodes was applied just above one edge of the monolayer, and cells were stimulated with monophasic, 10-ms pulses at 2 Hz to determine CV and APD at 80% repolarization (APD80). In monolayers with IK1 heterogeneity, reentrant spiral waves were initiated by rapid pacing, and their inducibility, stability, and frequency were determined. All data are expressed as mean±SD and compared using the paired Student’s t test. A probability value of <0.05 was considered statistically significant.
Characterization of Nontransduced and Kir2.1 Gene-Modified Cultures
Immunohistochemistry against Kir2.1, cTnI, actin, and Cx43 and Hoechst nuclear staining were performed to characterize the morphology, composition, and levels of Kir2.1 and Cx43 expression in 6-day-old nontransduced, LV-Empty–transduced, LV-Kir2.1–transduced, and LV-Kir2.1AAA–transduced monolayers. Immunohistochemistry against Kir2.1 showed that whereas nontransduced (Figure 1A) and LV-Empty–transduced (Figure 1B) NRVMs had low levels of native Kir2.1 protein, Kir2.1-transduced (Figure 1C), and Kir2.1AAA-transduced (Figure 1D) NRVMs had increased levels of wild-type Kir2.1 and dominant-negative mutant, Kir2.1AAA, respectively. Immunostain images of cTnI, actin, and Cx43 in nontransduced and transduced monolayers (Figure I in the online data supplement) confirmed that in a given field of view, both nontransduced and transduced cultures were morphologically similar and had similar levels and distributions of gap junctional protein expression. Western blot (Figure 1E) and integrated pixel density analysis (Figure 1F) showed similar levels of tubulin and Cx43 expression in all groups and greatly increased (up to 17-fold) levels of Kir2.1 (wild-type and dominant-negative mutant in LV-Kir2.1 and LV-Kir2.1AAA, respectively) expression in Kir2.1 gene-modified groups compared with the nontransduced and LV-Empty–transduced groups.
Characterization of Single-Cell Electrophysiological Properties of Kir2.1-Transduced NRVMs
We performed whole-cell patch clamp on 6-day-old enhanced green fluorescent protein (eGFP)-transduced and Kir2.1 gene-modified NRVMs to assess their single-cell electrophysiological properties. Transduced NRVMs exhibited enhancement or suppression of IK1 with overexpression or suppression of Kir2.1, respectively. When compared with eGFP-transduced NRVMs (−41.7±2.6 pA/pF; n=6; Figure 2A), the average IK1 density at −100 mV was significantly larger in Kir2.1-overexpressed NRVMs (−432.5±12.7 pA/pF; n=6; P=8.2×10−11; Figure 2B) and significantly smaller in Kir2.1-suppressed NRVMs (−5±1.4 pA/pF; n=6; P=2.3×10−9; Figure 2C). Spontaneous APs were absent in Kir2.1-overexpressed NRVMs, and single APs could be triggered by a short depolarizing current stimulus. As reported by previous studies,19,20 with a greater and almost complete suppression of IK1, Kir2.1AAA-transduced NRVMs fired spontaneous APs resembling those of genuine pacemaker cells. Spontaneous APs were also observed in eGFP-transduced NRVMs. Representative APs observed in eGFP-transduced, Kir2.1-transduced, and Kir2.1AAA-transduced NRVMs are shown in supplemental Figure II. Changes in IK1 also significantly shortened APD at 90% repolarization (APD90) in the Kir2.1-overexpressed group (29.9±9.1 ms; n=6; P=1.2×10−8) and significantly prolonged APD90 in the Kir2.1-suppressed group (183.9±7.2 ms; n=6; P=1.2×10−8) compared with the eGFP-transduced control group (116.3±6.8 ms; n=6). The current/voltage (I-V) relationship of Kir2.1 gene-modified and eGFP-transduced NRVMs is shown in Figure 2D. As shown in the inset, overexpression of Kir2.1 also boosted outward IK1, whereas suppression of Kir2.1 reduced outward IK1 when compared with the eGFP-transduced control group (at +30 mV, 76.4±5.3 pA/pF; P=1.2×10−7; n=6; versus 0.8±0.3 pA/pF; P=1.2×10−6; n=6; versus 6.8±0.9 pA/pF; n=6; in overexpressed, suppressed, and control groups, respectively). As expected, Kir2.1-overexpressed NRVMs showed a significantly hyperpolarized RMP (−79.3±0.7 mV; n=3; P=0.0013; Figure 2E), whereas Kir2.1AAA-overexpressed NRVMs had a depolarized maximum diastolic potential (−65±2.9 mV; n=3; P=0.0381) compared with eGFP-transduced NRVMs (−73.2±0.3 mV; n=3). The dV/dtmax for APs of Kir2.1-overexpressed NRVMs was significantly higher (260±27 V/sec; n=3; P=0.001) and that of Kir2.1-suppressed NRVMs was significantly lower (23.5±9 V/sec; n=3; P=0.003) when compared with control NRVMs (123±18 V/sec; n=3; Figure 2F).
Characterization of Tissue Electrophysiological Properties of Kir2.1-Transduced Monolayers
The tissue electrophysiological properties of uniformly Kir2.1 gene-modified monolayers significantly differed from those of nontransduced and LV-Empty–transduced monolayers. Representative isochrone maps for impulse propagation at 2-Hz pacing rate (Figure 3A) showed that APs propagated through nontransduced and LV-Empty–transduced monolayers with similar CV values of 19.1±0.5 cm/sec (n=7) and 18.9±0.7 cm/sec (n=7; P=0.07; compared with nontransduced control), respectively. However, APs propagated with increased CV (27.9±1.1 cm/sec; n=7; P=2×10−5; compared with nontransduced control) in Kir2.1-overexpressed monolayers and decreased CV (12.2±0.7 cm/sec; n=7; P=5×10−6; compared with nontransduced control) in Kir2.1-suppressed monolayers. Our findings of CV in Kir2.1-modified NRVM monolayers are consistent with our previous study17 and with our findings of increased dV/dtmax with Kir2.1 overexpression (Figure 2F). The suggestion of increased sodium channel availability and enhanced excitability with IK1 upregulation has also been previously reported.13 AP waveforms obtained from mapping (Figure 3B) showed normal APD80 values in nontransduced (174±6 ms; n=7) and LV-Empty–transduced (169±7 ms; n=7; P=0.27; compared with nontransduced control) monolayers, significantly abbreviated APD80 values in LV-Kir2.1–transduced monolayers (70±5 ms; n=7; P=9×10−7; compared with nontransduced control) and significantly prolonged APD80 values in LV-Kir2.1AAA–transduced monolayers (200±9 ms; n=7; P=2.8×10−4; compared with nontransduced control). Our findings of APD80 changes in Kir2.1-modified NRVM monolayers are consistent with previous findings20 and confirm that IK1 regulates the duration of the AP in these cells. Average CV and APD80 values for each group are summarized in Figure 3C and 3D. The average normalized upstroke velocity of propagating APs at a 2-Hz pacing rate in LV-Empty–transduced, Kir2.1-overexpressed, and Kir2.1-suppressed monolayers (n=7 each) were 3.23±0.42 (P=0.72), 5.36±0.43 (P=2.1×10−8), and 1.93±0.47 (P=2.4×10−7), respectively, when compared with that of nontransduced monolayers (3.11±0.62; n=7). Finally, in nontransduced and LV-Empty–transduced monolayers, successful 1:1 capture occurred up to a pacing rate of 4.5±0.3 Hz (n=7), whereas Kir2.1-overexpressed and Kir2.1-suppressed monolayers had a maximum capture rate of 9±0.5 Hz (n=7) and 3±0.3 Hz (n=7), respectively (Figure 3E). Importantly, reentry could not be induced in nontransduced and uniformly transduced monolayers even at a very high pacing rate (for example, 9 Hz for Kir2.1-overexpressed monolayers).
Development of an In Vitro Tissue Model of NRVMs With Heterogeneous IK1 Expression
Transduced NRVMs from a first day of cell isolation were spatially localized within a clear boundary defined by the stenciling technique (Figure 4A). Nontransduced NRVMs from a successive day of cell isolation were added to the whole coverslip and grown under normal culture conditions for an additional 5-day period (total of 6 days for original NRVMs) to develop an in vitro model of cardiac myocytes with a spatially localized functional heterogeneity (Figure 4B). Over a prolonged culture period of 6 days and beyond, nontransduced and transduced NRVMs microscopically coupled well without any discernible structural heterogeneities (Figure 4C). To assess the percentage of nontransduced NRVMs in the transduced central region, we transduced NRVMs from the first day of isolation with LV-eGFP and labeled nontransduced NRVMs from the second day of isolation with CellTracker Red CMTPX. Fluorescent images of the interface region showed that eGFP-transduced NRVMs were sharply confined within the circular boundary initially set by the stenciling procedure (Figure 4D). The nontransduced NRVMs attached to the outside of the gene-modified region and formed a monolayer, although some could be found in the transduced region. Collectively, the green and red channel images of the interface region confirmed the ability of the stenciling technique to create a confluent monolayer of nontransduced NRVMs with a central region of transduced heterogeneity (Figure 4E). Images from inside the gene-modified region (Figure 4F) showed that the central island consisted mostly of transduced NRVMs (80.9±1.8% of cell-covered area as measured from 3 monolayers with 1 field of view per monolayer).
Impulse Propagation in Monolayers With Regions of Kir2.1 Gene Modification
In monolayers with LV-Empty–transduced islands, at 2-Hz pacing rate, APs propagated through the islands with CV values similar to those of the rest of the monolayer (19.3±0.5 cm/sec in islands; n=9; P=0.32 and 19.6±0.4 cm/sec in the surrounding nontransduced region; n=9), as seen with an unperturbed linear wavefront in the direction of impulse propagation (Figure 5A, left column). As expected, APs propagated with increased CV (25.6±1.8 cm/sec; n=9; P=5.3×10−10; compared with 19.4±0.4 cm/sec in the nontransduced region; n=9) in Kir2.1-overexpressed islands and decreased CV (12.7±2.3 cm/sec; n=9; P=4.3×10−10; compared with 19.3±0.6 cm/sec in the nontransduced region; n=9) in Kir2.1-suppressed islands, as seen with the respective convex and concave curvature of the propagating wavefronts (Figure 5B and 5C, left column). Representative isopotential maps of AP propagation in monolayers with LV-Empty–transduced or Kir2.1 gene-modified islands are shown in supplemental Figure III. In LV-Empty–transduced islands, the APD80 values were similar to those of nontransduced regions (169±8 ms; n=9; P=0.12 and 175±8 ms; n=9, respectively). However, abbreviated APD80 (71±6 ms; n=9; P=6.6×10−14; compared with 168±7 ms in the nontransduced region; n=9) and prolonged APD80 (210±7 ms; n=9; P=4.1×10−6; compared with 172±4 ms in the nontransduced region; n=9) were seen in Kir2.1-overexpressed and Kir2.1-suppressed islands, respectively (Figure 5A through 5C, right column). The CV and APD80 values of LV-Empty, Kir2.1-overexpressed, Kir2.1-suppressed, and nontransduced regions were not statistically different (P>0.05 for all cases) from those observed for these groups in uniformly transduced monolayers.
Dynamics of Induced Reentrant Spiral Waves in Monolayers With Heterogeneous IK1 Expression
We next studied the dynamics of an induced spiral wave in monolayers with altered IK1. Rapid pacing failed to induce reentrant waves in monolayers with nontransduced and LV-Empty–transduced NRVM islands (n=9 each). However, reentry was successfully initiated in the 2 groups of monolayers with islands of heterogeneous expression of IK1. But the dynamics of reentry varied greatly between the 2 groups. Reentrant waves were anchored to islands of Kir2.1 overexpression (Figure 6A), remained stable for a prolonged period of time (more than 2 hours), and had a frequency of 9±1 Hz (n=9), as shown by the optical recordings from a representative recording site (Figure 6B). When the tip of the induced spiral wave was tracked over 3 successive cycles, it maintained an almost circular trajectory inside the region of Kir2.1 overexpression (Figure 6C). The reentrant wave propagated with a CV of 6.4±0.2 cm/sec (n=9) in the transduced region at a distance of 2 mm from the wavetip and a CV of 16.3±0.4 cm/sec (n=9) in the nontransduced region at a distance of 6 mm from the wavetip. After 1 minute of superfusion (includes wash-in time and time allowed for the solution to mix in the chamber) with Tyrode’s solution containing 50 μmol/L Ba2+, which selectively blocks IK1 currents, the frequency of reentry gradually decreased to 7±1 Hz (n=9). The stability of the spiral wave was also disturbed, as reflected by a transition from a circular wavetip pattern to a meandering pattern of almost the same size. The previously measured reentry CVs in the transduced and nontransduced regions of the monolayer decreased to 5.1±0.2 cm/sec (n=9) and 14.1±0.5 cm/sec (n=9), respectively. The changes in the dynamics of the reentry after 1 minute of Ba2+ superfusion are summarized in supplemental Figure IV. After 3 minutes of Ba2+ superfusion, the stability of the reentry was heavily lost (Figure 6D), and the rotation frequency decreased further to 5±1 Hz (n=9; Figure 6E). The trajectory of the spiral wavetip also followed a meandering pattern much larger in size than before (Figure 6F). The reentry CV in the transduced and nontransduced regions decreased further to 3.2±0.2 cm/sec (n=9) and 11.7±0.2 cm/sec (n=9), respectively. On continued Ba2+ superfusion, the reentry terminated within the next 2 to 4 minutes.
Similar to a previous report,19 dominant-negative suppression of Kir2.1 gave rise to spontaneous activity (6 of 9 monolayers). This activity emerged from inside the transduced region to the rest of the monolayer (Figure 7A) at a frequency of 1.5±0.5 Hz (n=6). Rapid pacing of monolayers with islands of Kir2.1 suppression successfully initiated reentrant waves (Figure 7B), which were quasi-stable. The spiral wave moved around the island with a frequency of 3±1 Hz (n=9). Shortly after initiation (within 3 to 5 minutes), it detached from the island and terminated (3 of 9 monolayers), transitioned into a sustained complex of coupled spiral waves (4 of 9 monolayers; Figure 7C), or transitioned into a 2-armed spiral wave (2 of 9 monolayers; Figure 7D) that rearranged itself into a figure-of-eight reentry (Figure 7E), which drifted away from the island and terminated. Thus, Kir2.1-suppressed NRVM islands stabilized reentry but with a lower frequency and shorter duration compared with Kir2.1-overexpressed NRVM islands.
In a final set of experiments, monolayers with an inverse pattern of IK1 heterogeneity were developed with nontransduced NRVM islands in the center and Kir2.1 gene-modified NRVMs on the outside. Nontransduced NRVM islands with LV-Empty–transduced NRVMs on the outside were used as controls. Although reentry could not be induced in control cultures, it was successfully initiated in monolayers with an inverse pattern of Kir2.1 overexpression or suppression. However, the dynamics of the reentry varied greatly between the 2 groups. With Kir2.1 overexpression, reentrant spiral waves were stable and persisted for more than 2 hours (Figure 8A) and had a frequency of 7±0.5 Hz (n=9). The frequency of the reentry was slower than the 9±1 Hz seen in monolayers with central islands of Kir2.1 overexpression. The tip of the spiral wave followed an almost circular pattern in the region of Kir2.1 overexpression over the course of 3 successive cycles (Figure 8B). In an opposite fashion, with Kir2.1 suppression, the induced spiral wave was unstable and terminated within 5 minutes after initiation (Figure 8C). The reentry had a frequency of 3±0.5 Hz (n=9) similar to the 3±1 Hz seen in monolayers with central islands of Kir2.1 suppression, and its tip followed a meandering pattern (Figure 8D). Thus, in the case of inverse heterogeneity, altered IK1 expression also enhanced the genesis of spiral waves compared with uniformly transduced monolayers, with greater stability for IK1 overexpression compared with IK1 suppression.
Thus, with heterogeneous Kir2.1 expression, spiral waves are consistently observed to be more stable with Kir2.1 overexpression than with Kir2.1 suppression, whether altered Kir2.1 expression occurs in the island or in the surrounding region (for the inverse pattern). These observations support the notion that decreased head–tail interaction secondary to shortened APD contributes to the stability of reentrant waves.
Studies have previously suggested the importance of IK1 in VF dynamics by relating the stability of high-frequency rotors to the levels of outward component of IK1.12 Increased IK1 during atrial fibrillation at hyperpolarizing potentials is also considered to be an important factor for the maintenance of the arrhythmia.21 In transgenic animals, the role of IK1 upregulation in the frequency and stability of rotors has been studied.13 However, in such transgenic hearts, there was reported to be cell-to-cell variability in the expression of IK1,16 and the effects of such heterogeneities on arrhythmia dynamics are unknown. Hence, in the present study, using a combination of tissue engineering, somatic gene transfer, immunohistochemistry, Western blot, whole-cell patch clamp, and optical voltage-mapping techniques, we developed a novel, in vitro model of cardiac tissue with a spatially localized IK1 heterogeneity and characterized the electrophysiology and dynamics of reentrant spiral waves in this tissue model. In comparing such a tissue model against transgenic animals, there is the advantage (and disadvantage) of not having compensatory mechanisms coming into play for the gain or loss of function of proteins of interest. Our study demonstrates the capacity of IK1, when heterogeneously expressed, to initiate and maintain a high-frequency spiral wave in cultured NRVM monolayers. Hence, it may not be simply the increase in IK1 that contributes to increased stability of spiral waves13 but also the heterogeneity in IK1 expression and the subsequently large IK1 differences. The faster rotation rates and persistence of the rotor have been previously attributed to the increased outward component of IK1.5 Our study provides new insight on how spiral wave anchoring by IK1 can predispose the heart to fatal cardiac arrhythmias. Hence, altering Kir2.1 expression levels and modulating IK1 currents may lead to alternative and potentially effective antiarrhythmic approaches.
IK1 Regulates Excitability and Electrophysiological Properties of Cardiac Tissue
In this study, we demonstrated that overexpression of Kir2.1 significantly increased IK1 density, whereas dominant-negative suppression of Kir2.1 significantly decreased IK1. Also, upregulation of IK1 significantly hyperpolarized RMP, increased dV/dtmax and shortened duration of the AP. These findings are consistent with the role of IK1 in regulating sodium channel availability and excitability of cardiac tissue.13 As expected, suppression of IK1 significantly depolarized RMP, decreased dV/dtmax, and prolonged duration of the AP, indicative of reduced channel availability.
The secondary effects of modulation of IK1 on cardiac electrophysiological tissue properties, namely APD and CV, were then characterized. We chose bipolar line stimulation because with flat excitation wavefronts, the effects of wavefront curvature on CV can be eliminated. Furthermore, the bipolar electrodes concentrate the stimulation current in the gap between the electrodes and hence avoid distant stimulation. In contrast, the convex excitation wavefronts produced by a point stimulus propagate slower than flat excitation wavefronts and facilitate the formation of reentrant waves.
We observed increased CV in monolayers with uniform Kir2.1 overexpression and decreased CV in monolayers with Kir2.1 suppression. Because CV depends on gap junction coupling and excitability, the absence of changes in Cx43 levels between nontransduced, LV-Empty–transduced and Kir2.1 gene-modified monolayers together with the observed changes in dV/dtmax support the notion that CV changes following Kir2.1 gene modification are mediated by associated changes in RMP and sodium channel availability. Another factor that may alter upstroke velocity and CV in the case of IK1 modification is suppression or exaggeration of phase IV pacemaker depolarization as a consequence of altered outward currents in the threshold voltage range. As expected, Kir2.1 overexpression abbreviated APD80, whereas Kir2.1 suppression prolonged APD80. Similar to a previous report,20 IK1 suppression resulted in prolonged APD and spontaneous activity in islands of Kir2.1AAA-transduced NRVMs. In Kir2.1AAA-transduced monolayers, APD, and thus the effective refractory period, is significantly prolonged with a much shorter excitable gap, suggesting there could be incomplete recovery of the sodium channel at high stimulation rates, in addition to the reduction in sodium channel availability owing to the reduction of RMP.
IK1 Regulates Cardiac Reentry Dynamics
Sustained arrhythmias have been previously induced in cultured monolayers of NRVMs using a rapid pacing protocol that relies on preexisting heterogeneity in cellular or tissue properties (eg, excitability, refractory period, or anisotropy) to cause the formation of a wavebreak. In this study, the uniformity of tissue properties was such that rapid pacing failed to induce stable reentry in nontransduced monolayers. Furthermore, rapid pacing failed to induce reentry in uniformly transduced monolayers and in monolayers with islands of nontransduced or LV-Empty–transduced NRVMs. On the other hand, the presence of sharp CV and APD differences in monolayers with islands of Kir2.1 overexpression or suppression, or with the inverse patterns, promoted reentry initiation. Interestingly, whereas reentry could be induced at pacing rates of ≈5 Hz in monolayers with islands of Kir2.1 overexpression, and at 5.6 Hz in monolayers with the inverse pattern, no reentry could be induced even at 9 Hz in monolayers with uniform overexpression of Kir2.1. Similarly, reentry could be induced in monolayers with heterogeneous but not uniform Kir2.1 suppression, although the reentrant waves were less stable than those observed with heterogeneous Kir2.1 overexpression. The findings of our study demonstrate that the uniform overexpression of Kir2.1 does not necessarily create a medium that is prone to reentry formation and that it is IK1 heterogeneity that enhances the genesis of stable spiral waves. Following Ba2+ perfusion of monolayers containing islands of heterogeneous Kir2.1 expression, IK1 is blocked in both the transduced and nontransduced regions of the monolayer, lowering the level of IK1, abolishing IK1 heterogeneity, and transforming the monolayer into a homogeneous medium. Hence, the spiral wave meanders, loses stability, and eventually terminates by drifting off the edge of the monolayer.
In our study, both the differences and level of IK1 density seem sufficient to account for many aspects of VF dynamics. Nevertheless, we cannot discount the involvement of other outward currents in the manifestation of cardiac arrhythmias. Recently, increased IKs has been shown to enhance conduction block and wavebreak formation by means of postpolarization refractoriness.22 However, simulations have predicted that IK1 has a greater effect on rotor frequency than other potassium currents such as Iss, Ito, and IKslow.13 Thus, IK1 is an important regulator of spiral wave frequency and stability because of its role in governing the excitation threshold, as well as the terminal phase of repolarization.13
Our results from perturbations of IK1 in the central island indicate that changes in rotation rate of the spiral wave can occur from perturbations in IK1 expression solely near the spiral tip without changes in the arms of the spiral wave, unlike the case with transgenic animal experiments.13 Also, more stable spiral waves are consistently observed with Kir2.1 overexpression than with Kir2.1 suppression, whether altered Kir2.1 expression occurs in the island or in the surrounding region. In conclusion, our study provides new experimental evidence for the contribution of IK1 to cardiac arrhythmogenesis and emphasizes the potential importance of IK1 as an antiarrhythmic target.
We thank Peihong Dong and Seth Weinberg for assistance with molecular biology and mapping data analysis, respectively.
Sources of Funding
This work was supported by American Heart Association Predoctoral Fellowship 0715212U (to R.B.S.); National Heart, Lung, and Blood Institute grant P0-1 HL77180 (to G.G.H.; D.A. Kass, principal investigator), Fondation Leducq (to E.M.), and NIH grant HL66239 (to L.T.).
↵*Both authors contributed equally to this work.
This manuscript was sent to Harry Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received April 28, 2008; revision received December 10, 2008; accepted December 17, 2008.
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