Role of apamin sensitive small conductance calcium-activated potassium currents in long term cardiac memory in rabbits

Abstract

Background

Apamin-sensitive small conductance calcium-activated K current (I_KAS) is upregulated during ventricular pacing and masks short-term cardiac memory (CM).

Objective

To determine the role of I_KAS in long-term CM.

Methods

CM was created with 3-5 weeks of ventricular pacing and defined by a flat or inverted T-wave off pacing. Epicardial optical mapping was performed in both paced and normal ventricles. Action potential duration (APD₈₀) was determined during RA pacing. Ventricular stability was tested before and after I_KAS blockade. Four paced hearts and 4 normal hearts were used for western blotting and histology.

Results

There were no significant differences in either the echocardiographic parameters or in fibrosis levels between groups. Apamin induced more APD₈₀ prolongation in CM than in normal ventricles (9.6% [8.8%-10.5%] vs 3.1% [1.9%-4.3%], p<0.001). Apamin significantly lengthend the APD₈₀ in the CM model at late activation sites, indicating significant I_KAS upregulation at those sites. The CM model also had altered Ca²⁺ handling as the 50% Ca²⁺ transient duration and amplitude were increased at distal sites compared to a proximal site (near the pacing site). After apamin, the CM model had increased VF inducibility (paced vs control, 33/40 (82.5%) vs 7/20 (35%) P<0.001), and longer VF durations (124 vs 26 seconds, P<0.001).

Conclusions

Chronic ventricular pacing increases Ca²⁺ transients at late activation sites which activates I_KAS to maintain repolarization reserve. I_KAS blockade increases VF vulnerability in chronically paced rabbit ventricles.

Introduction

Unnecessary right ventricular (RV) pacing may cause cardiomyopathy in patients with preserved left ventricular systolic function¹ and increased morbidity and mortality in patients with reduced ejection fraction with implantable cardioverter-defibrillators.² The mechanisms of the detrimental effects of ventricular pacing remain incompletely understood. Ventricular pacing is known to produce both short and long term cardiac memory (CM) characterized by T-wave changes associated with altered activation sequence.³ Recent studies have shown that ventricular pacing is associated with cytosolic calcium (Ca_i) accumulation at late activation sites.⁴ The increased Ca_i can cause significant changes of cardiac repolarization, including increased sodium-calcium exchange current (I_NCX) that tend to prolong the action potential duration (APD) and promote afterdepolarizations.⁵ On the other hand, the increased Ca_i could also activate apamin sensitive small conductance Ca²⁺ activated K (SK) current (I_KAS), which helps to reduce the APD and preserve the repolarization reserve.⁶ Because of the presence of these two opposing effects, ventricular pacing can cause transient APD prolongation at late activation sites leading to a positive APD-activation time (AT) relationship (short term CM) within 5-min of pacing.⁷ Continued pacing for an hour or more can normalize the APD-AT relationship due to I_KAS upregulation in late activation site.⁶ Therefore, short term CM is transient and is well compensated by the upregulation of I_KAS. However, the mechanism of long term CM remains unclear. We hypothesize that I_KAS is upregulated at late activation sites during chronic ventricular pacing in rabbit ventricles but is of insufficient magnitude to suppress the manifestation of CM. Apamin, which blocks I_KAS, aggravates APD prolongation at the late activation sites, reduces repolarization reserve and increases ventricular vulnerability to fibrillation. The purpose of the present study was to test these hypotheses.

Methods

The study protocol was approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine and the Methodist Research Institute, Indianapolis, Indiana, and conformed to the regulations for humane care and treatment of animals established by the NIH. A total of 29 female New Zealand white rabbits (3.5-5.0 kg) were used in this study. Among them, 20 were used for chronically ventricular pacing and 9 normal rabbit hearts that were not paced were used as controls. A detailed methods section is included in the Online Supplement.

Pacing-Induced long-term CM and Optical Mapping

Ventricular epicardial pacing of either the right ventricle (n=9) or the left ventricle (n=11) was used to induce long-term CM.⁸ Ventricles were paced at 270 beats per min (bpm) for 3-5 weeks. Each week, the T-wave from ECG Lead II was assessed. Chronic CM was defined by either a flat or inverted T-wave (Figure 1). Echocardiography was performed both before the pacemaker implantation and at follow up. Optical mapping was performed on both paced hearts and normal hearts of both the RV and LV (Online Supplement Figure 1) as previously described.⁸ Excised hearts were Langendorff perfused at 25 to 35 mL/min with Tyrode’s solution and stained with Rhod-2 AM (1.48 μmol/L) for Ca_i and RH237 (10 μmol/L) for V_m mapping. Contractility was blocked with blebbistatin (15 – 20 μmol/L). To prevent induction of short term CM the hearts remained in normal sinus rhythm except for specific programmed stimulation during optical recordings.⁶

Figure 1.

Evolution of CM over 28 days of ventricular pacing (VP). Depicted is lead II from a rabbit heart with RV pacing and with LV pacing (A) showing the T-wave at baseline, and a flat or inverted T-wave after pacing was discontinued on day 28. (B) Amount of fibrosis from Masson’s trichrome staining of ventricular tissue samples from structurally normal hearts (control) and chronically paced hearts. Summary data shows that there was no significant difference in fibrosis levels between the CM model and the control group.

Protocol I – Electrophysiological Effects of Cardiac Memory

The CM model was created in 7 hearts with RV pacing and 3 hearts with LV pacing to determine if LV pacing showed the same characteristics as RV paced hearts. Five normal hearts were used as controls. The ventricular APD₈₀ was determined in each heart by pacing the right atrial appendage at 300 ms, and optical maps were acquired after at least 30 paced beats. To determine the role of I_KAS, apamin (100 nmol/L) was then added to the perfusate and the protocol was repeated after 30 minutes. At that concentration, apamin is a highly specific I_KAS blocker.⁹ After radiofrequency ablation of the atrioventricular (AV) node, pacing was performed from the RV apex. A S1/S2/S3 (short/long/short) pacing protocol (S1 30 beats with S1-S1 300 ms, a long S1-S2 of 1000 ms or 2000ms and a S2-S3 starting from 300 ms and gradually shortened to the ventricular effective refractory period (ERP)) was used to simulate the ECG characteristics that initiates early after-depolarizations (EADs) and torsades de pointes (TdP) ventricular tachycardia in the clinical setting.¹⁰ Finally, a dynamic ventricular pacing protocol was used to determine arrhythmia inducibility. If VF was not induced, burst pacing (PCL 50 ms, pacing duration 10 s) was used to induce VF. Any VF that occurred was allowed to continue for 180 s before a defibrillation attempt was made. After the return to sinus rhythm, the heart was allowed to rest for 1 min before continuing the pacing protocol. VF induction was attempted 4 times in each heart. Phase singularities (PS) were used to quantify VF characteristics.¹¹

Protocol II – Effects of Cardiac Memory on Arrhythmia Inducibility

In protocol I, all arrhythmia inducibility data was acquired after apamin infusion. To determine the effects of CM on arrhythmia inducibility and the role of I_KAS, 3 additional hearts with chronic LV pacing were used to acquire data on ventricular stability at baseline and after apamin infusion. In each heart, AV nodal ablation was performed, and to determine the inducibility of any EADs and VF, ventricular pacing was performed as described in protocol I. Any ventricular arrhythmias that resulted from the pacing were noted and then apamin (100 nmol/L) was added to the perfusate and the protocol was repeated after 30 min.

Western Blotting

Among the 3 different subtypes of SK channels, SK2 is known to be the most sensitive to apamin and contribute the most to I_KAS.¹² To determine the protein expression of SK2 channels within the myocardial tissue, Western blotting was performed on tissue acquired from sites distal and proximal to the chronic pacing site in 4 chronically paced hearts and from similar areas in 4 normal hearts.¹³

Histology

For histology, ventricular tissue from 4 normal hearts and 4 chronically paced hearts was embedded in paraffin, sectioned and stained using Masson’s trichrome stain. The collagen content was quantified by identifying and counting the number of blue-staining pixels as a percentage of the total tissue area using digital photomicrographs in Adobe Photoshop CS6 software.

Quantitative PCR

Quantitive polymerase chain reaction (PCR) was used to determine the expression of transcripts encoding kcnn2 (SK2) and kcnn3 (SK3) in tissue acquired from sites distal and proximal to the chronic pacing site in 3 chronically paced hearts.

Statistical Analysis

Data are presented as mean and 95% confidence interval (CI). Paired Student t tests were used to compare variables measured at baseline and after apamin infusion. An independent-sample t test was used to compare variables measured between groups. A one way ANOVA was used to compare SK2 protein expression between different groups, and a paired t-test was used to compared RNA levels between the proximal and distal sites. Categorical parameters between groups comparing VF vulnerability were compared by either a chi-square test or the Fisher exact test. A 2-sided P value of ≤0.05 was considered statistically significant.

Results

All rabbits survived the chronic pacing protocol. Long term CM was assessed by T-wave changes in the ECG (flat or inverted T-wave after pacing). As shown in Figure 1, after 28 days of ventricular pacing, the ECG shows a T-wave that changed to flat or inverted in both RV and LV paced hearts. There were no significant differences in echocardiographic based measurements of LV size and function between baseline and after chronic pacing. Ventricles demonstrated an LV end-diastolic dimension of 12.2 mm [95% CI, 11.3–13.3] vs 12.1 mm [95% CI,11.4–12.8], for 3-5 weeks pacing and baseline, respectively; P=0.649), end-systolic dimension (8.2 mm [95% CI, 7.3–9.1] vs 8.0 mm [95% CI, 7.4–8.7]; P=0.637), fractional shortening (32.9% [95% CI, 29.4–36.4] vs 32.4% [95% CI, 28.6–36.2]; P=0.81) and ejection fraction (69.3%[95% CI, 64.5–74.1] vs 69.6% [95% CI, 64.3–74.9] P=0.907). From histological analysis shown in Figure 1B, there was no difference in the amount of fibrosis between chronically paced hearts and normal hearts.

Effects of Apamin on the APD in CM

Figure 2A shows APD₈₀ maps (measured during RA pacing at 300 ms PCL) before and after apamin infusion in normal ventricles, in RV paced ventricles and in LV paced ventricles studied in Protocol 1. Corresponding optical tracing of action potentials from a single representative pixel from the site marked with an “A” are also shown in the figure. At baseline, the APD₈₀ distant from the pacing site had no or minimal prolongation while the APD₈₀ closer to the pacing site was prolonged. After apamin, the APD₈₀ was prolonged throughout the mapped region, indicating significant I_KAS upregulation at sites remote from pacing (arrows) in both RV and LV paced ventricles. There was little or no APD₈₀ prolongation in normal ventricles. Figures 2B–2D summarize the effects of chronic pacing on APD₈₀ distribution at an atrial pacing cycle length (PCL) of 300 ms in all ventricles studied in Protocol I. As the summary data shows, apamin induced more APD₈₀ prolongation in paced ventricles than in normal ventricles (paced vs Normal, 9.6% [8.8%-10.5%] vs 3.1% [1.9%-4.3%], p<0.001, Figure 2D), with no differences between RV and LV paced ventricles. APD heterogeneity has been shown to be an important factor that contributes to increased vulnerability to ventricular arrhythmias.¹⁴ Figure 2C shows that after apamin the standard deviation (SD) of APD₈₀ significantly increased at 300 PCL in CM hearts, but not in normal hearts.

Figure 2. APD distribution before and after I_KAS blockade by apamin.

Optical V_m mapping was performed during RA pacing at a 300 ms PCL. A shows APD tracings (first row) from site A marked in the color maps below. Example APD₈₀ maps are shown at baseline (second row), after apamin (100 nmol/L) infusion (third row) and the ΔAPD₈₀ between baseline and apamin (4^th row). Arrows point to a significant lengthening of the APD₈₀ at the late activation site in both RV and LV paced ventricles. Asterisks indicate the sites of chronic pacing. B – Summary APD₈₀ data showing that apamin prolonged the APD₈₀ in normal and CM ventricles. C – Standard Deviation (SD) of APD₈₀ is larger in CM ventricles compared to normal ventricles both before and after apamin infusion. Apamin significantly incresed the SD of APD₈₀ in CM ventricles, but not in normal ventricles. D – APD₈₀ was significantly prolonged after apamin infusion in CM ventricles compared to normal ventricles.

Characteristics of Ca²⁺ distribution in the CM model

We investigated the effects of the chronic pacing on the Ca²⁺ cycling characteristics by comparing the 50% Ca²⁺ transient duration (CaTD₅₀). Sample Ca²⁺ signals from a normal heart and a paced heart are shown in figure 3. Overlapping the signals demonstrates that both RV and LV paced hearts have a longer CaTD₅₀ than normal hearts. Summary data in panel C shows a significant increase in the CaTD₅₀ in paced hearts compared to normal hearts. Ca²⁺ signals recorded with optical mapping also revealed a spatial gradient of the Ca²⁺ transients in the paced hearts. In both the RV and LV chronically paced hearts, Ca²⁺ fluorescence was increased at distal sites (site remote from the pacing site) compared to proximal sites (near the pacing site). However, this relationship did not exist in the normal ventricles. Sample normalized Ca²⁺ signals (F/F0) from both the proximal and distal locations are shown in Figure 4. For all chronically paced ventricles, the F/F0 measured was 1.070 (N=10, CI 1.052-1.088) near the chronic pacing site (early activation site), which was significantly lower than the F/F0 (1.103, N=10, CI 1.084-1.123, P<0.001) measured at the opposite site of the ventricle (late activation sites).

Figure 3. CaTD₅₀ changes in normal and chronically paced hearts.

A – CaTD₅₀ maps from optical mapping of rabbit hearts from both control and paced groups. Maps were recorded during atrial pacing (PCL of 300ms). Asterisks indicate the site of chronic ventricular pacing prior to explantation of the heart. B – Example Ca_i transient recordings show that the decay was prolonged in the CM model. Summary data is shown in Panel C.

Figure 4.

Effects of chronic pacing on Ca_i fluorescence transients in the CM model (A – RV pacing, B – LV pacing) as compared to control (C). All optical tracings were obtained during atrial pacing. Asterisks indicate the site of chronic ventricular pacing prior to explantation of the heart. Ca²⁺ fluorescence levels were increased at late activation sites compared to early activation sites near the location of chronic pacing in CM model (Panels A and B). However, the same was not observed in Panel C (normal control).

Chronic ventricular pacing is proarrhythmic

In protocol I, a dynamic ventricular pacing protocol was used to determine arrhythmia inducibility between normal and paced hearts. For all hearts studied, after apamin, paced ventricles developed APD alternans (Figure 5A) at a longer PCL than the control group (232ms [CI, 194–270] vs 180 [95% CI, 160–200 P=0.006] (Figure 5B). No spontaneous arrhythmias were observed during the study. In 10 paced hearts and 5 normal hearts, VF induction was attempted in each heart 4 times. VF vulnerability increased (paced vs control, 33/40(82.5%) vs 7/20(35%) P<0.001) (Figure 5C), with a longer duration in paced hearts compared to control hearts (124 vs 26 seconds, P<0.001) (Fig 5D).

Figure 5. Ventricular vulnerability to fibrillation after I_KAS blockade in normal and chronically paced ventricles (Protocol I).

A - Representative traces of V_m alternans in the CM model after apamin infusion. Corresponding AT and APD maps from two consecutive ventricular activations are shown. B - Apamin prolonged the PCL threshold of 2:2 alternans. C - VF inducibility was tested 4 times in each heart. VF was able to be induced in all 10 paced ventricles, and in 3 of the 5 normal ventricles. D - After apamin infusion, VF duration was longer in chronic paced hearts compared to control hearts. The pacing sites during the mapping study were the same as that during chronic ventricular pacing . AT – activation time.

In protocol II, 3 additional hearts were studied to investigate ventricular vulnerability to fibrillation at baseline and after apamin infusion. Figure 6A shows representative examples of optical mapping traces at baseline and after addition of apamin demonstrating a significant prolongation of the APD₈₀ at a PCL of 1000 ms after apamin infusion. At baseline, only one of the three paced hearts developed a TdP arrhythmia. However, after apamin infusion, all of the three paced hearts developed a TdP arrhythmia during short-long-short pacing. An example of TdP induced with short-long-short pacing is shown in Figure 6B. In addition, the site of earliest activation of the TdP occurred at the sites farthest from the chronic pacing site (Figure 6C). In these 3 CM hearts, there were 5 VF episodes at baseline and 11 episodes after apamin infusion. Examples of phase maps during VF with marked phase singularities (black arrows) at baseline and after apamin infusion is shown in Figure 7A and 7B. Figure 7C summarizes the number of phase singularities in all hearts studied in Protocol II. There was a significant increase of phase singularities after apamin.

Figure 6. Optical mapping of pacing-induced Tdp in Protocol II (AV block).

A – APD₈₀ maps and example signals at baseline and after apamin infusion in the CM model. Shown is that the APD is significantly prolonged at a PCL of 1000 ms. B - EAD beat induced TdP during short-long-short pacing after apamin infusion (300-2000-220ms). C - The earliest activation site of an TdP occurred at a site distal from the chronic pacing site. Red arrows point to the origin of the TdP. An AT map is shown along with a fluorescence map which shows an area with light red color (V_m gradient change), which is the earliest activation site of the earliest TdP beat. An APD₈₀ map shows that the earliest activation site correlated to the longest APD. The pacing during optical mapping was performed from the same site as during chronic LV pacing .

Figure 7.

Effect of I_KAS blockade on the wave breaks during VF in the CM model. Panels A and B show consecutive phase maps sampled at 20 ms during VF at baseline and after apamin infusion. Phase singularities (PS) are indicated by black arrowheads. A corresponding optical recording of VF from the site indicated by the asterisk is also shown. Note that the numbers of PS were significantly increased after apamin infusion. Summary data is shown in Panel C.

SK2 Protein and RNA Expression

Western blotting was performed in 4 normal hearts and 4 chronically paced hearts (2 RV pacing and 2 LV pacing). As the results in Figure 8 show, there was no significant difference in the SK2 protein expression between normal and chronically paced hearts. However, qPCR analysis showed that there was a significant increase in both SK2 (0.20 [95% CI, 0.08-0.31] vs. 0.27 [95% CI, 0.21-0.32], p=0.048) and SK3 (0.21 [95% CI, 0.13-0.28] vs. 0.24 [95% CI, 0.13-0.34], p=0.045) levels at the distal site compared to the proximal site.

Figure 8.

Protein and mRNA analyses. (A) Western blot analyses of the SK2 protein levels from chronically paced ventricles and in normal controls. (B) There was no significant difference in the SK2 protein levels in paced ventricles compared to normal ventricles or between proximal and distal sites. (C) RNA levels show a significant increase in SK2 and SK3 expression at the distal site compared to the proximal site.

Discussion

This study has shown that CM develops with persistent ventricular pacing, which results in a repolarization gradient across the epicardial surface during atrial pacing, with the longest APD₈₀ near the chronic pacing site and the shortest APD₈₀ at the distal sites within the mapping field of view. Infusion of I_KAS inhibitor apamin significantly lengthened the APD₈₀ with the greatest increase of APD₈₀ at the locations distal from the chronic pacing site. In addition, an increase in the Ca²⁺ transients was also observed at these distal sites. Putting this information together, chronic pacing resulted in Ca_i accumulation and a robust activation of I_KAS at late activation sites. Because of the increased I_KAS, the late activation sites had a shorter APD than the early activation sites. This abnormal distribution of APD persisted when ventricular pacing was turned off, resulting in longer APD₈₀ at the apical pacing sites than at the base. This abnormal distribution of APD₈₀ results in abnormal patterns of T-waves during sinus or atrially paced rhythms (chronic CM).

I_KAS blockade and ventricular vulnerability to fibrillation

In paced ventricles, inhibiting I_KAS increased VF vulnerability and the incidence of TdP induced with EADs. These data support that I_KAS plays a significant compensatory role in CM and protects against arrhythmia initiation. Several commonly used drugs, including amiodarone¹⁵ and ondansetron,¹⁶ are known to block I_KAS. Whether or not I_KAS inhibition increases arrhythmia risk for pacemaker patients deserves further investigation.

Mechanisms of cardiac memory

CM is characterized by a change in T-wave morphology resulting from either electrophysiological or structural remodeling induced by either ventricular pacing or myocardial stretch.^{17, 18} Varying degrees of CM has been shown after minutes to hours of pacing (producing “short-term” CM) and after 2 to 5 weeks of pacing (“long-term” CM). Two weeks of pacing is required to achieve a steady-state change in the T-wave. Molecular mechanisms of CM have been extensively reviewed recently.^19,20 Short-term CM that is observed within minutes of ventricular pacing is thought to occur from modulation and modification of angiotensin II which has been shown to regulate I_to. Long-term CM, seen after longer periods of ventricular pacing and is longer lasting (days to weeks), includes alterations in AT1 receptors, stretch-activated receptors,^{21, 22} connexins^{23, 24} and in ion channels specifically I_to, I_kr, and I_Ca,L which results in an altered repolarization gradient. The mechanisms underlying this ion channel remodeling revolves around changes in gene transcription and protein synthesis.^19,20 This study is the first to show that in addition to the currents mentioned above, I_KAS may also play a role in long-term CM.

Clinical Implications

This study provided new insights into the mechanisms of CM. The importance of I_KAS in human ventricular repolarization is supported by a previous study that showed apamin prolonged the APD in the failing human ventricular myocardium.²⁵ In CM models, we have observed an increased APD heterogeneity, steepened maximal slope of APDR, increased PCL threshold for alternans and increased spatially discordant APD alternans after I_KAS inhibition, factors which increase vulnerability to ventricular arrhythmias. These findings suggest drugs that block I_KAS may be unsafe for patients with chronic ventricular pacing. A second clinical implication is that I_KAS upregulation helps to shorten the APD₈₀ and counteract the detrimental effects of Ca²⁺ accumulation at sites distal from pacing. Therefore, only a minority of patients develop pacing-induced cardiomyopathy after prolonged periods of RV pacing.¹

Study Limitations

We only studied the acute effects of apamin in an ex vivo perfused heart model. We did not investigate the chronic administration of a SK channel blocker in an ambulatory cardiac memory model. In addition, we did not investigate SK1 or SK3 protein levels as a reliable antibody is not available for rabbit hearts.

Conclusions

Chronic ventricular pacing increases Ca_i at late activation sites which helps to activate I_KAS to shorten the APD and maintain repolarization reserve. Inhibiting I_KAS significantly lengthens the APD at late activation sites and increases VF vulnerability. I_KAS blockade may contribute to the underlying mechanism of sudden cardiac death in patients with permanent ventricular pacing.