Showa Univ J Med Sci 19(3), 123 135, September 2007

Original Effects of Nifekalant Injected into the Pericardial Space on the Transmural Dispersion of in Pig

Hiroyuki ITo, Taku ASANO, Youichi KOBAYASHI, Tatsuya ONUKI, Fumito MwosHI, Taka-aki MATSUYAMA, Yoshino MINOURA, Norikazu WATANABE, Mitsuharu KAWAMURA, Kaoru TANNO and Takashl KATAGIRI

Abstract: Transmural dispersion of repolarization (TDR) has been implicated

in the onset of ventricular . We investigated the effects of nifekalant (NIF) injected into the pericardial space on TDR and in

pig. We injected 50 or 100 mg of NIF into the pericardial space of 11 pigs, and measured the effective refractory period (ERP) between the endocardial

and epicardial myocardial cells, as well as QT time and QT peak-end as an

index of TDR and T waveforms, respectively. TDR decreased from 56•} 10 msec to 44•}8 msec (P < 0.01), 5.1 min after injection of 100 mg of NIF,

although the QTc did not change. At a later time, QTc increased from 457 •}

44 msec before injection to 540•}49 msec (P < 0.01) and TDR recovered to

the control level. When 50 mg of NIF was injected, the T wave amplitude

decreased from 0.433 •}0.301 mV before injection to 0.107•}0.192 mV (P < 0.01) at 10 min after injection ; 100 mg of NIF caused the T wave amplitude to decrease more rapidly, reaching a negative value at 4.5 min after injection.

Injecting NIF into the pericardial space altered ERP, QT, and T waveform,

and also decreased TDR.

Key words : transmural dispersion of repolarization, electrophysiological study, injection into pericardial space

Introduction

Recent studies showed that the dispersion of repolarization time from epicardial cells to endocardial cells (transmural dispersion of repolarization ; TDR) has an impact on the electrocardiogram (ECG) changes associated with and the onset of ventricular arrhythmia 1-3). Short QT syndrome (SQTS) is a novel genetic inherited disorder that occurs in individuals with a structurally intact but an increased susceptibility to ventricular and sudden death. The SQTS subtype, SQTS, has a unique ECG phenotype characterized by asymmetrical T waves, as well as a defect in the gene coding for the inwardly rectifying Kir 2.1 (1K1) channel4, 5) Nifekalant (NIF) blocks IKr to extend the action potential duration (APD) in a frequency- dependent manner, which in turn increases QT time and effective refractory period (ERP). Recently, NIF was used as a first-line drug in cardiopulmonary resuscitation6,7) When Third Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8600, Japan. 124 Hiroyuki ITO, et al administered intravenously, NIF increases TDR, induces phase 2 re-entry, and causes 8, 9), possibly because of the increased ERP in the endocardial myocardium. NIF administrated into the epicardial space might therefore shorten TDR, although, to the best of our knowledge, this has not been tested 10). This study investigated the effects of NIF injected into the pericardial space of pigs on ERP of the endocardial and epicardial myocardial cells, as well as on QT time, TDR, and T waveforms, as measured by surface ECG. Subjects and Methods

Subjects included eleven female pigs aged 3-4 months and weighing 31•}2 kg. The following drug preparations were prepared : an injection containing 50 mg or 100 mg of NIF and physiological saline at 37•Ž as the control. Each drug preparation was dissolved in physiological saline at 37•Ž (total 5 ml) immediately before injection. Pigs were placed in the supine position and arterial oxygen saturation was monitored.

Anesthesia was induced with 5 mg / kg of xylazine and 5 mg / kg of ketamine intravenously, and maintained with inhaled 1.1 % •` 2.7 % isoflurane. Pigs were intubated and ventilated with N20 and O2 (1:1) . The Seldinger method was employed to puncture the right internal carotid vein and both femoral veins and arteries, and a 7 or 8 French sheath was inserted into each artery and vein. Through the sheath placed in the right internal carotid vein and the left and right femoral veins, a multiple electrode catheter (Boston Scientific-EP

Technologies) was placed : an 8-electrode catheter in the high right atrium, an 8-electrode catheter in the right cardiac apex, and a 10-electrode catheter in the coronary sinus. In addition, from the left margin of the sternum in the fourth intercostal space, pericardiocen- tesis was performed using an epidural needle (17G). Small amounts of contrast media were injected through the needle as it was advanced into the pericardial space under fluoroscopy guidance. When the pericardial space was thought to be reached, a small bolus of contrast media was injected, and this was confirmed by diffusion of contrast media throughout the pericardial space. A guidewire was then passed through the needle, over which the 8F sheath was advanced. Through the sheath placed in the epicardial space, a 7 Fr electrode catheter was inserted and placed on the anterior wall side of the left ventricular epicardium under fluoroscopy guidance in order to perform the electrophysiological study (Fig. 1).

Methods for drug injection

Drug injection followed the schedule shown in Fig. 1. As a control, 10 ml of physiologi- cal saline (37•Ž) was injected into the pericardial space over a 30-s period, and EPS was performed starting at 1 min after the injection. The pig was monitored for 5 min after the injection, and the drug solution was eliminated. Next, after a break of about 10 min, either 50 or 100 mg of NIF was then dissolved in 37•Ž physiological saline to bring the volume to 5 ml, and this was then injected into the epicardial space over a 30-s period.

ERP measurement was initiated at 1 min after the injection. ERP was measured from both the epicardial and endocaldial side at the same time. The pig was monitored for 30 min after the injection, and the drug solution was then eliminated. During this period, surface ECG was continuously monitored. The same procedures were performed on each of the eleven pigs. Nifekalant Decrease TDR in Pericardial Space 125

Fig. 1. Drug injection protocol and catheter location. After pericardiocentesis, catheters were located at the position shown in the photo.

Observation items and test methods 1. Electrophysiological test From each electrode catheter placed in the left ventricular apical and epicar- dium, continuous stimulation with a basic cycle length (BCL) of either 400 or 500 ms was applied, and the minimal output with which the left ventricle responded was measured as the threshold value. The intensity of ventricular stimulation was twice the threshold value, and further experiments were conducted. After delivering stimulation seven times at 400 or 500 ms BCL by the early stimulation method, the shortest interval for QRS waves in relation to single early simulation with a shortened interval was measured as ERP. Measurement was started 1 min after the injec- tion and required about 5 min to complete. In three pigs, ERP was also measured at 8 min and 18-20 min after the injection. 2. ECG Using Cardio Lab (GE Medical), surface 12-lead and intracardiac ECG was digitally recorded continuously. Paper speed was set at 50 mm / s with a gain of 20 mm / mV. RR interval (msec), QT time (msec) and T wave amplitude (mV) in V1 lead were measured on the Cardio Lab screen every 30 sec from immediately before drug injection to 5 min after injection, every 1 min from 5 to 10 min after injection, every 2 min from 10 to 20 min after injection, and every 3 min from 20 to 30 min after injection. As shown in Fig. 2, QT peak (QTp) was defined as the distance from the starting point of the QRS complex to the peak of the T wave, serving as an indicator for APD of myocardium of the 126 Hiroyuki ITO, et al

Fig. 2. QRS measurement methods in V1 QT peak (QTp) was defined as the distance from the starting point of the QRS complex to the peak of the T wave. QTend (QTe) was defined as the distance from the starting point of the QRS complex to the endpoint of the T wave, and was measured by the tangential method, serving as an indicator for the APD of M cells distributed in the middle myocardial layer. QTpeak-end (QTpe) was defined as the distance from the peak of the T wave to the endpoint of the T wave, serving as an indicator for TDR. epicardial side. QTend (QTe) was defined as the distance from the starting point of the QRS complex to the endpoint of the T wave, and was measured by the tangential method, serving as an indicator for the APD of M cells distributed in the middle myocardial layer. QTpeak-end (QTpe) was defined as the distance from the peak of the T wave to the endpoint of the T wave, serving as an indicator for TDR 1).

After correcting using Budget's formula (QTc = QT / RR112), each parameter (QTcp,

QTce and QTcpe) was expressed as mean •} standard deviation (SD) and was then plotted. Comparison was made among four time points : (1) before injection, (2) maximum change in QTcpe after injection, (3) maximum change in QTce, and (4) end of observation.

3. Plasma concentration Blood samples were collected at 1, 5, 10, 15, 20 and 30 min after injecting NIF, and plasma concentration was measured by HPLC (Mitsubishi BCL). 4. Ethics The current study was performed according to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institute of Health. The study protocol was reviewed by the Committee of the Ethics on Animal Experiments of Showa University.

Analysis methods Chronological changes in ERP and ECG parameters (compared with those before drug injection) were analyzed, and stratified comparison (low-dose group, high-dose group, epicar- dium group and endocardium group) was carried out using 2-sample t-test or paired t-test. Nifekalant Decrease TDR in Pericardial Space 127

QTc (peak,end,peak-end) Physiological saline

QTc(peak,end,peak-end)

Fig. 3. QTc (Physiologicalsaline and Nifekalant) QTce : Corrected QT peak, QTce : Corrected QTend, and QTcpe : Corrected QT peak-end. No clear changes were seen in QTce or QTcpe before and after injection of physiologicalsaline (p = NS). At 9.7 min after Nifekalant injection (100mg), both QTce and QTce increased significantlyand remained high at 30 min after injection.

These analyses were performed with the Stat View 5J program (SAS Institute, Cary, NC). Significantdifferences were defined as P < 0.05, while P < 0.1 was considered to indicate a trend. 128 Hiroyuki ITO, et al

Results

1. QT time, QTc peak-end (QTcpe) in V1 Injection of physiological saline produced no change in QTce, Qtcp, or QTcpe before and after injection (p = NS) (Fig. 3-1).

When 50 mg of NW was injected, no significant changes were seen in either QTce or

QTce in the early phase (5.8 min), but QTcpe tended to decrease from 91•}14 ms to 55 •} 23 ms (P < 0.1). Both QTce and QTce then tended to change from 506 •} 53 ms and 414 •}

39 ms (before injection) to 536 •} 62 ms and 464 •} 61 ms (8.3 min after injection), respectively

(P < 0.1), while QTcpe recovered simultaneously. At 30 min after injection, both QTce

and QTce had recovered from 536 •} 62 ms and 464 •} 61 ms (8.3 min after injection) to 513 •}

70 ms and 422 •} 83 ms respectively, restoring QTcpe to control levels (Fig. 3-2).

Injection of 100 mg of NW induced no significant changes in either QTce or QTce in the early phase (5.1 mm), but QTcpe significantly decreased from 56•}10 ms to 44 •} 8 ms (P < 0.01). In addition, both QTce and QTce significantly changed from 457 •} 44 ms and 401 •}

42 ms (before injection) to 540 •} 49 ms and 485 •} 40 ms (9.7 min after injection), respectively

(P < 0.01), but neither recovered, even at 30 min after injection, while QTcpe recovered simultaneously (Fig. 3-2). 2. Amplitude and morphology of T wave in V1

Injection of physiological saline produced no clear change in T wave amplitude (p =

NS) (Fig. 4-i). Injection of 50 mg of NW decreased the T wave amplitude from 0.433 •}

0.301 mV before injection to 0.107 •} 0.192 mV at 10 min after injection (P < 0.01). When

100 mg of NW was injected, T wave amplitude rapidly decreased from 0.25 •} 0.10 mV before injection to -0.01 •} 0.11 mV at 3.5 min after injection (P < 0.01). The T wave amplitude tended to have a negative value at 4.5 min after injection, decreasing from 0.25 •}0.10 mV before injection to - 0.051 •} 0.10 mV (Fig. 4-2 and 5). 3. ERP

Injection of physiological saline produced no clear change in ERP (p = NS) (Fig. 6).

Injection of 50 mg of NW increased ERP on the epicardial side significantly from 285 •}

34 ms to 312 •} 31 ms (P < 0.01). However, ERP on the endocardial side showed no clear change, increasing slightly from 292 •} 24 ms to 308 •} 28 ms (p = NS). Injection of 100 mg of

NW significantly increased ERP on both the endocardial and epicardial sides from 270 •} 35 ms to 322 •} 28 ms and from 275 •} 34 ms to 321 •} 30 ms, respectively (P < 0.01) (Fig. 6).

Fig. 7 shows the chronological changes in ERP. Prior to the NW injection, ERP on the epicardial side was 285 •} 34 ms. It increased to 312 •} 31 ms at 1 min after injection (P <

0.01), and remained high for 20 min. On the endocardial side, ERP before injection was

293 •} 25 ms, increasing to 333 •} 33 ms at 8 min after injection, which was 1 min later than the increase on the epicardial side (P < 0.01). 4. Plasma concentration

Fig. 8 shows shifts in the plasma concentration of NW following intravenous administra- tion into humans and pericardial injection to pigs. Five minutes after injection of 50 mg of

NW into the pericardial space of pig, the plasma concentration of NW was 225 •} 107 ng / ml, which was comparable to that at 5 min after intravenous injection (0.2 mg / kg) into humans. When 100 mg of NW was injected, the plasma concentration of NW at 5 min after injection was 419•}273 ng / ml, which was comparable to that at 5 min after intravenous injection Nifekalant Decrease TDR in Pericardial Space 129

T wave amplitude (mV) Physiological Saline

T wave amplitude (mV) Nifekalant

Fig. 4. Time course of T wave amplitude in V1 lead (Physiological saline and Nifekalant) Physiological saline did not cause any significant change in T wave amplitude. After Nifekalant injection, T wave amplitude decreased gradually, and at 100 mg, T wave amplitude had a negative value in some cases. 130 Hiroyuki ITO, et al

Time course of T wave morphology (Nifekalant100mg)

Fig. 5. Time course of T wave morphology (Nifekalant 100mg) When 50 mg of Nifekalant was injected, T wave amplitude gradually decreased (4 min after injection) (P< 0.01), while 100mg of Nifekalant decreased T wave anplitude (3 min after injec- tion) (P < 0.01) and showed a tendencg toward a negative value in a dose-dependentmanner.

(0.3mg / kg) into human. These plasma concentrations indicate no significant arrhythmo- genecity.

Discussion The main findings of this study are as follows. NIF injected into the pericardial space of pig decreased QTcpe (TDR indicator) in the early phase, after which it recovered. This indicated that NW was effective only on the epicardial side in the early phase of 1-8 min after injection. When NW was injected into the epicardium, the shape of T waves trended downward in a dose-dependent manner. 1. QT time Injection of NW into the pericardial space increased both QTcp and Qtce in the present study. Shimizu and Antzelevitch1)examined sections of arterially perfused left ventricular myocardium, and found that QTpeak, as assessed by surface ECG, represents the APD of epicardial myocardium, while QTend represents the APD of M cells (mid-myocardial cells in the middle myocardial layer). In the present study, NW shortened QTcpe without changing QTce for up to 5.8 min, suggesting that NW only increased the APD of epicar- dium. Furthermore, QTce was also increased 9.7 min later, and APD tended to recover. These results showed that injection of NIF into the pericardial space expresses its pharma- cological effects over time from the epicardial myocardium to the endocardial myocardium, and the QT-increasing effects of the present study do not contradict the results of studies using intravenous injection. Nifekalant Decrease TDR in Pericardial Space 131

ERP(msec)

Fig. 6. ERP ERP (Physiological saline) ; Following physiological saline injection, no significant differences were seen in epicardial and endocardial ERP. ERP (Nifekalant) ; Following 50 mg Nifekalant injection, ERP was significantly increased only on the epicardial side. Therefore it was thought that at 1-5 min after the injection, the pharmacological effects of Nifekalant were only apparent on the epicardial side. But with a dose of 100 mg, infiltration to the endocardial side was faster, thus negating the difference between the epicardial and endocardial sides.

2. TDR Shimizu and Antzelevitch also proposed QTcpe (peak-end) as a TDR indicator 1), involved in the ECG changes associated with Brugada syndrome and in the onset of ventricular arrhythmia. In the present study, NW decreased QTcpe without increasing Qtend soon after injection, and as a result, NW only increased QT on the epicardial side. This suggested that NIF decreases TDR soon after injection into the pericardial space, thus preventing phase 2 re-entry. These findings have clinical implications in suggesting that NW injected 132 Hiroyuki ITO, et al

Fig. 7. Time course of ERP With Nifekalant, ERP increased significantly on the epicardial side at 1 min after injection, and it remained high for 20 min after injection. On the endocardial side, a clear increase was seen at 8 min after injection.

into epicardial side of the pericardial space might help to prevent the onset of electrical storm in Brugada syndrome. Recent reports have linked epicardial catheter mapping and ablation of ventricular 12). Future administration of drugs to the epicardial side might therefore be expected, for example, during open-heart surgery or catheter mapping. 3. Amplitude and morphology of T wave Many cases of idiopathic ventricular (VF) in younger individuals with structurally normal have been documented 13). Some of what was thought to be idiopathic are now defined as specific clinical entities, such as long QT syndrome, Brugada syndrome, cat- echolaminergic (VT), and SQTS 14). SQTS is characterized by a high incidence of , sudden cardiac death and supraventricular arrhythmias, short refractory period, and inducibility of VF during programmed electrical stimulation 15). Among the sub- types of SQTS, SQT3 (KCNSJ2) is characterized by an asymmetrical T wave with a rapidly descending limb, and peculiar tall and narrow T-wave patterns 4). Brugada syndrome is characterized by ST-segment elevation in the right precordial leads (V1-3) and an episode of VF in the absence of structural heart disease 16). In an experimental Brugada syndrome model using arterially perfused right ventricular myocardial sections, pinaci- dil, a K + channel opener, enhanced ST-segment elevation 17). Late r' waves and ST elevation were also reported to become prominent just before a VF episode in Brugada syndrome 18). Nifekalant Decrease TDR in Pericardial Space 133

Time course of plasma concentration of Nifekalant

Fig. 8. Plasma concentration of Nifekalant following intravenous administration (humans) or pericardial injection (pigs) Even when large doses of Nifekalant were injected into the epicardium, large amounts of the drug were not transferred to the blood circulation. Furthermore, at these plasma concentrations, there was no significant arrhythmogenecity.

However, the mechanism responsible for ST elevation in Brugada syndrome remains unclear. In this study, injection of NW into the epicardium caused the T wave shape to trend downwards in all cases. At the high dose of NW, the T wave even reached a negative value. Our results provide some clues to a likely mechanism for ST depression. 4. ERP NIF injection into the pericardial space in this study increased ERP. This does not contradict the ERP-increasing action of intravenous NIF. ERP was also measured with time following the pericardial NIF injection, and pharmacological effects were seen earlier in the epicardium than in the endocardium. The results indicated that such effects of NW are only apparent on the epicardial side early after injection, thus injection of this drug into the pericardial space seems more effective than intravenous injection. At a dose of 50 mg, NW markedly increased ERP on the epicardial side alone, while 100 mg NIF increased ERP on both the epicardial and endocardial sides. These findings suggested that 100 mg of NW caused the infiltration from the epicardial side into the endo- cardial side to be excessively rapid, thus making no difference between the epicardial and endocardial sides (Fig. 6). We propose approximately 50 mg (1.6 mg / kg) of NW as the optimal dose. 134 Hiroyuki ITO, et al

5. Plasma concentration Five minutes after injecting 100 mg of NIF into the pericardial space of pigs or intrave- nously administering 0.3 mg / kg NIF to patients, plasma concentrations of the drug in both cases was of the order of 400 ng / ml. This result demonstrated that NIF injected into the pericardial space moved quickly from the epicardial side to the endocardial side, and affected electrophysiological activity comparably with the intravenous injection. In addition, these findings indicated that even large doses of NIF injected into the epicardium do not translate to high levels in the blood circulation.

Conclusions Injection of NIF into the pericardial space brought about specific changes in ERP, QT, and T waveform, while it decreased TDR.

Limitations

The present study had certain limitations that should be noted, despite it suggesting the usefulness of epicardial administrating of NW in cases of ventricular arrhythmia. First, the number of subjects was limited. Second, it has not been confirmed that the clinical effects of NW in pigs will be similar in human patients. We are planning studies to overcome these limitations and enhance the significance of the findings.

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[Received January 19, 2007: Accepted February 1, 2007]