Quinidine Depresses the Transmural Electrical Heterogeneity of Transient Outward Potassium Current of the Right Ventricular Outflow Tract Free Wall

Background— Electrical heterogeneity of the right ventricular outflow tract (RVOT) is regarded as one of the main electrophysiological substrates for Brugada syndrome. Recently quinidine has shown efficacy in patients with Brugada syndrome due to its ability to inhibit potassium current especially 4-aminopyridine–sensitive, non-Ca2+ -dependent transient outward potassium current (Ito). However, much less is known on how extent quinidine in clinical therapeutic concentration range can inhibit this kind of electrical heterogeneity of RVOT Ito. Methods and Results— Single RVOT free wall epicardial (Epi) cell, midmyocardial (M) cell and endocarcial (Endo) cells were used for whole-cell voltage clamping and Ito was recorded at 37°C, 0.2 Hz depolarization pulse. Evident Ito tranmural heterogeneity existed in RVOT free wall. Under the condition of baseline, of 10 μM quinidine perfusion 5 minutes (mins), and of 10 μM quinidine perfusion 7–10 mins, from 0 mV to 70 mV the whole transmural average Ito values of RVOT free wall were 10.2 pA/pF, 5.5 pA/pF and 3.5 pA/pF, respectively (between groups, P< 0.01). The inhibitory percentage of 10 μM quinidine at 5 mins and 7–10 mins steady-state level on the the whole Ito transmural heterogeneity of RVOT free wall were 46.3%±6% and 66.5%±11%, respectively. Conclusions— There exists a robust Ito transmural electrical heterogeneity in RVOT free wall and quinidine in clinical therapeutic concentration can depress this kind of heterogeneity effectively.


INTRODuCTION
It's estimated every year there are 200,000-400,000 persons suffered from sudden cardiac death (SCD) in the USA only. 1 SCD is the leading cause for middle-aged death and it forms a big challenge to public health all over the world. 1 SCD most commonly occurs as a consequence of acute myocardial ischemia or develops with identifiable substrate such as scar because of previous myocardial infarction, cardiomyopathy, or hypertrophy. Despite extensive noninvasive and invasive evaluation, SCD remains unexplained without evidence of structural heart disease in about 10% of individuals surviving SCD. It is now recognized that inherited electrophysiological abnormalities, termed primary electrical diseases, are the common underlying cause of these unexplained cardiac arrests. These conditions include Wolff-Parkinson White syndrome, long QT syndrome, Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT), short QT syndrome and idiopathic ventricular fibrillation (IVF). 2 As a kind of ion channelopathy paradigms and monogenic mutant leading to SCD, Long QT syndrome, Brugada syndrome and short QT syndrome provide us with some important keys to understand the electrophysiological mechanism of SCD. [3][4][5][6][7][8][9] Recently, more and more experimental studies have suggested that Brugada syndrome and some IVFs originate from the right ventricular outflow tract (RVOT), and regional transmural heterogeneity is one of the main electrophysiological substrates of malignant arrhythmias caused by Brugada syndrome, short QT syndrome and some IVFs. [10][11][12][13] Prominent 4-aminopyridine-sensitive, non-Ca 2+ -depen dent transient outward potassium current (Ito) of right ventricular epicardial (Epi) cell and midmyocardial (M) cell is one of the most prominent and important manifestations of the right ventricular transmural electrical heterogeneity. 7 The prominent Ito mediated action potential notch in Epi cells and M cells, but not that in endocardial (Endo) cells, create transmural voltage gradients and thus cause ST-segment elevation. Right ventricular Ito is an important ionic basis for repolarization "spike and dome" morphology, phase 2 "all or none" repolarization, action potential plateau heterogeneous loss and phase 2 reentry. On the basis of phase 2 reentry, ventricular tachycardia (VT) or ventricular fibrillation (VF) can be precipitated easily by a short coupling extrasystole. This is one of the most important electrophysiological mechanisms which are responsible for SCD of Brugada syndrome and some IVF victims. 7,12 Because the presence of a prominent Ito in the RVOT is regarded as an important link to the mechanism underlying the Brugada syndrome, the important rational approach to therapy, regardless of the ionic or genetic basis for the disease, is to partially inhibit Ito. [14][15][16][17] As a robust potassium current inhitor especially Ito inhibor, quinidine is effective in suppressing wedge arrhythmogenesis models of the Brugada syndrome. 12 Clinically, although implantation of an automatic implantable cardioverter defibrillator (ICD) is the recommended first choice for most of the Brugada syndrome, short QT syndrome and IVF patients, antiarrhythmic therapy with quinidine aimed at rebalancing the currents activity during phase 1 of the right ventricular action potential is used to abort electrical storms, as an adjunct to device therapy, and as an alternative to device therapy when use of an ICD is not possible. [14][15][16][17] Today, more and more evidence has shown the excellent long-term reproducibility of the electrophysiological efficacy of quinidine in patients with Brugada syndrome, short QT syndrome and some IVFs. [18][19][20][21][22][23] However, limited information can be available on how effective of quinidine on restoring the Ito electrical homogeneity of the RVOT. In this study, we examined the Ito electrical heterogeneity of the RVOT free wall and the effect of clinical therapeutic concentration quinidine on this kind of Ito electrical heterogeneity.

Prepared solutions
The following solutions were prepared as previously described by our laboratory: 24

Isolating canine right ventricular outflow tract free wall cells
The methods and protocols used in canine were approved by Capital Medical University Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, revised 1985). Cells were prepared as previously described by our laboratory and Paul G. A. Volders'experience. 24,25 Briefly, 15 mongrel dogs of either sex (about 18±4 kg each) were anesthetized with pentobarbital sodium (30 mg/kg). Their hearts were excised quickly and placed into Ca 2+ -free buffer solution gassed with 95% O 2 -5% CO 2 . The right coronary artery branches, which supply blood to the free wall of the RVOT, were then cannulated and perfused with Ca 2+ -free solution at a rate of 60 ml/min. After about 15-30 mins of enzymatic solution reperfusion, samples of Epi cells (<1.5 mm from the epicardial surface), M cells (2-5 mm from the epicardial surface), and Endo cells (<1.5 mm from the endocardial surface) were dissected with a fine dermatome while carefully avoiding contamination between the three cell types. Cell samples were gently agitated, filtered, and washed, and then stored at room temperature for later use.

Ito recording
Ito was recorded using whole cell patch-clamp techniques with an EPC-9 amplifier, and controlled by Pulse+Pulsefit 8.31 software (Heka, Germany) as previously described by our laboratory. 24 Briefly, cells were allowed to adhere to the bottom of the chamber for 3-5 mins, and then were continuously superfused with extra cellular solution at a rate of 2 ml/min. Only relaxed quiescent cells displaying prominent cross striations were used. All experiments were performed at 37°C. The resistance of all borosilicate glass pipette tips measured in extracellular solution was 2-4 MΩ when filled with pipette solution. After whole-cell configuration established, the series resistance and the slow capacitance were compensated automatically by Pulse 8.31 software to prevent large voltage errors. The activated and inactivated voltage-clamp pulse protocols are illustrated in Figures 1-5. The peak Ito amplitudes were measured as peak amplitudes from baseline minus steady-state values at the end of the test pulses. To analyze 4-AP-sensitive, non-calcium dependent Ito, it was necessary to minimize other temporarily superimposed currents. In our study, the L-type calcium current was blocked with 150 µmol/L CdCl 2 added to the extracellular solution. T-type calcium currents, which are relatively small in canine ventricular cells, were also reduced with extracellular CdCl 2 solution. Calcium-activated chloride currents were suppressed by blocking I Ca with CdCl 2 , and by using 10 mmol/L EGTA in the pipette solution. The fast sodium current was inactivated by using a 10-ms prepulse at -45 mV (Figures 1  and 2).

Drug
Quinidine sulfate (Beijing Municipal Chemical Plant, Beijing, China) was dissolved in the external solutions at a concentration of 10 mM and diluted to the final concentration.

Statistical analysis
All data were expressed as mean±SEM. and analyzed with SPSS 11.5. Differences were statistically analyzed by the t test or ANOVA. P<0.05 was considered to be statistically significant.

Baseline Ito heterogeneity between three cell types of the RVOT free wall
At different depolarizing voltage levels, Ito density (pA/pF) were the biggest in RVOT free wall Epi cells (n=14), followed by M cells (n=14) and, finally, Endo cells (n=12). Ito sometimes was absent in Endo cells (Table 1, Figure 1).
At 37°C, using 0.2 Hz depolarization pulses at 0 mV and at 70 mV, the average peak Ito density in RVOT Epi cells was bigger than that in M cells (

DISCuSSION
The data above clearly demonstrated that there exists strong transmural Ito electrical heterogeneity in RVOT free wall. Quinidine at the concentration of 10 µM, which was in clinical therapeutic range, possesses the ability to depress the strong transmural Ito electrical heterogeneity of RVOT free wall effectively.

Electrical heterogeneity of three types of canine right ventricular cells
Using arterially perfused wedges of canine right ventricle (RV), by simultaneously recording transmembrane action potentials from 2 epicardial and 1 endocardial sites, together with unipolar electrograms and a transmural Electrocardiograph (ECG), Yan et al. demonstrated that depression or loss of the action potential dome in RV epicardium creates a transmural voltage gradient that may be responsible for the ST-segment elevation observed in the Brugada syndrome and other syndromes exhibiting similar ECG manifestations. Their results also demonstrated that extrasystolic activity due to phase 2 reentry can arise in the intact wall of the canine RV and serve as the trigger for VT/VF. Their data also pointed to Ito blocker (4-aminopyridine, quinidine) as an effective pharmacological treatment. 12 However, for the RVOT free wall, which was regarded as the seedbeds of Brugada syndrome and some IVF, the information on trnasmural electrical heterogeneity, especially Ito trnasmural electrical heterogeneity of RVOT free wall was very limited. Our data above clearly indicated that there exist marked Ito electrical heterogeneity and transmural wall voltage gradients between Epi cells and Endo cells, and between M cells and Endo cells in the canine RVOT free wall. So, our results not only confirm the existence of potent Ito electrical heterogeneity in the RVOT free wall, but also provide further convincing quantitative evidence supporting the hypothesized mechanism for J wave formation, J point elevation, and ST-segment elevation on ECG of right precordial leads.

Clinical relevance
The electrical heterogeneity of the right ventricle has a close relationship with certain right ventricular malignant arrhythmias found in patients with Brugada syndrome and some IVFs.
Recently, more and more studies have verified in most rodents, canines, and humans, there is obvious Ito electrical heterogeneity in the ventricles during repolarization (phase 1). The different Ito distributions and transmural voltage gradients are among the most prominent and most important manifestations of right ventricular electrical heterogeneity, and one of the important ionic bases for the "spike and dome" action potential differences between the three cell types, 1), may be one of the main electrophysiological mechanisms for sudden cardiac death in patients with Brugada syndrome and other conditions, [3][4][5][6][7][8]10,12 although, there are still some controversies. 26 Under the supporting of the electrical heterogeneity and the repolarization disorder theory above, recently quinidine, as an adjunct to ICD therapy, has been used in treatment of IVF, Brugada syndrome and short QT syndrome successfully. More and more papers reported the profit of antiarrhythmic therapy with quinidine as an adjunct to ICD therapy to abort electrical storms, or the profit of quinidine as an alternative to device therapy when use of an ICD is not possible. 14,15,[17][18][19][20][21][22][23] However, limited information can be available on the Ito transmural electrical heterogeneity of RVOT free wall, one important seedbed for some clinical malignant arrhythmia and much less is known how extent quinidine in clinical therapeutic concentration range can depress the Ito transmural electrical heterogeneity coming from RVOT free wall. So, our results primarily answered the critical problems above and provided a further evidence for quinidine in treatment of some IVF, Brugada syndrome and short QT syndrome.
Based on the results of our study, we can conclude that there exists a robust Ito transmural electrical heterogeneity in RVOT free wall and quinidine in clinical therapeutic concentration can depress this kind of heterogeneity effectively.

ACKNOWLEDGEMENT
The authors would like to express thanks to Mrs. Xiulan Liu for her assistance in dog's care and cardiac cell isolation.   and for J point elevation, J wave formation and ST-segment elevation, action potential asymmetrical loss, "all or none" repolarization, and reentry into phase 2. VT or VF induced by phase 2 reentries, as a result of powerful transmural Ito electrical heterogeneity in the right ventricle during repolarization (phase