Effects of increasing ICa on spiral wave dynamics: Results from a 2-D experimental model

Poster 4 POSTER SESSION 4 Friday, May 6, 2005 Session Time: 9:00 a.m.–12:00 p.m. Presenter Available: 9:45 a.m.–10:45 a.m. Location: Exhibit Hall P4-1 BIOPHYSICAL PROPERTIES OF TWO TYPES OF ATP-SENSITIVE Kⴙ CHANNEL IN RAT VENTRICULAR MYOCYTES Adonis Zhi-Yang Wu, Ming Wei Lin, Ruey J. Sung and Sheng Nan Wu. National Cheng Kung University Medical College, Taiwan Republic of China, Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Taiwan Republic of China and National Cheng Kung University Medical College, Tainan, Taiwan Republic of China. The ATP-sensitive K⫹ (KATP) channels are known to provide the functional linkage between the electrical activity of the call membrane and metabolism. In molecular composition, previous study had shown that two types of inwardly rectifying K⫹ channel subunits (Kir6.1 & Kir6.2) and only a type of sulfonylurea receptors (SUR2A) that associated with Kir6.2 constituted the KATP channels in microsomal fractions of rat heart. In contrast, there is only one Kir6.1 KATP channel in smooth muscle. In this study, we provide evidence to show two types of KATP channels with different biophysical properties functionally expressed in rat ventricular myocytes. Using patch clamp technique on isolated rat ventricular myocytes, we found that single-channel conductances for the KATP channels are 56 and 21 pS, The kinetic properties, including open time and bursting kinetics, did not differ between two types of KATP channels. Dinitrophenol and rotenone could enhance the activity of these two types of KATP channels. Diazoxide only activated small-conductance KATP channel, while pinacidil stimulated both channels. Upon membrane excision, small-conductance KATP channels readily ran down, and further application of ATP restored channel activity. These channels are sensitive to inhibition by glibenclamide, in a similar magnitude. Additionally, western blotting revealed two types of Kir6.X channels, i.e., Kir6.1 and Kir6.2, in rat ventricular myocytes. These two types of KATP channels are related to functional activities in rat heart. P4-2 THE MODULATION OF ANGIOTENSIN II RECEPTOR TYPE 2 ON IONIC CURRENTS IN HUMAN ATRIAL MYOCYTES Chunbo Chen, Shulin Wu, Chunyu Deng, Yumei Xue and Weimin Qian. Guangdong Provincial Cardiovascular Institute, Guangzhou, China. Objective: To study the modulation of angiotensin II receptor type 2 (AT2) on ionic channel currents in human atrial myocytes. Methods: Right atrial appendages were obtained at the time of cardiac surgery and single myocytes were isolated by enzymatic dissociation with the chunk method. Whole-cell patch-clamp techniques were used to observe the effects of AT2 on L-type calcium current ( ICa,L), fast sodium current ( INa), transient outward potassium current ( Ito), inward rectifier potassium current ( IK1) and ultrarapid delayed rectifier potassium current ( IKur) in atrial myocytes from patients with sinus rhythm(SR) or chronic atrial fibrillation(⬎6months;AF). Results: The ionic current densities of ICa,L, INa and Ito in both SR and AF cells were not altered by the selective AT2 agonist CGP 42112(5 ␮mol/ L),respectively. At command potential of -120mV,CGP 42112(5 ␮mol/L) significantly increased IK1 densities by 20.5% (n⫽5/4,cells/patients; P⬍0.05) in SR cells and 29.1%(n⫽5/4;P⬍0.05) in AF cells. The selective AT2 antagonist PD 123319(1 ␮mol/L) completely inhibited the effects of CGP 42112,which had no direct effect on IK1 densities.At command potential of -120mV, CGP 42112(1,5,10␮mol/L) increased IK1 densities by 13.6%(n⫽5/3), 20.2% (n⫽6/4),46.1%(n⫽5/3) in SR cells and 18.4%(n⫽5/ 3),34.8%(n⫽6/4),50.1% (n⫽5/4) in AF cells, respectively, in a concentration-dependent manner.At command potential of ⫹50mV, CGP 42112(5

S215 ␮mol/L) significantly increased IKur densities by 22.2% (n⫽6/4;P⬍0.001) in SR cells and 28.4%(n⫽6/4;P⬍0.05) in AF cells.PD 123319 (1 ␮mol/L) completely inhibited the effects of CGP 42112, which had no direct effect on IKur densities.At command potential of ⫹50mV, CGP 42112 (1,5,10␮mol/L) increased IKur densities by 15.5% (n⫽8/4), 22.3% (n⫽8/ 5), 36.5% (n⫽7/5) in SR cells and 21.6% (n⫽5/4), 32.7% (n⫽6/4), 79.2% (n⫽5/4) in AF cells, respectively, in a concentration-dependent manner. Conclusions: Activation of AT2 increases IK1 and IKur densities, which may be a contributory factor to the induction and maintenance of AF.These findings indicate that AT2 is associated with ionic remodeling of AF in humans. P4-3 TUMOR NECROSIS FACTOR-〈 REGULATES CALCIUM HANDLING IN PULMONARY VEIN CARDIOMYOCYTES Yi-Jen Chen, MD, PhD, Yao-Chang Chen, ScD, MS, Shih-Lin Chang, MD, Cheng-I Lin, PhD and Shih-Ann Chen, MD. Taipei Medical University, Wan-Fang Hospital, Taipei, Taiwan Republic of China, National Defense Medical Center, Taipei, Taiwan Republic of China and Veterans General Hospital-Taipei, Taipei, Taiwan Republic of China. Background: The recent study showed that inflammation plays an important role in the genesis of atrial fibrillation, and those patients with atrial fibrillation showed a higher level of c-reactive protein compared to controls. Our previous study has shown that tumor necrosis factor-␣ (TNF-␣), a proinflammatory cytokine, may increase pulmonary vein (PV) arrhythmogenic activity with the increase of delayed afterdepolarization and transient inward currents. The purpose of this study is to investigate whether TNF-␣ regulates calcium homeostasis in PV cardiomyocytes and facilitates the PV arrhythmogenicity. Methods: Whole-cell patch clamp and indo-1 fluorimetric ratio technique were used to investigate the L-type calcium current, Na⫹-Ca2⫹ exchanger current and intracellular calcium in isolated single rabbit PV cardiomyocytes with and without (control) incubation of 25 ng/ml TNF-␣ for 7⬃10 hours. Results: TNF-␣-treated PV cardiomyocytes have a smaller L-type calcium current and larger Na⫹-Ca2⫹ exchanger current than control PV cardiomyocytes. Moreover, TNF-␣-treated PV cardiomyocytes (n⫽ 23) have a smaller intracellular calcium transient (R410/485, 0.15⫾0.01 versus 0.22⫾0.03, P⬍0.05) and larger diastolic intracellular calcium level (R410/ 485, 0.67⫾0.01 versus 0.61⫾0.02, P⬍0.05) than control PV cardiomyocytes (n⫽22). In addition, TNF-␣-treated PV cardiomyocytes have longer decay portion of calcium transient (Tau) than control PV cardiomyocytes (51⫾4 ms versus 32⫾4 ms, P⬍0.05). Conclusions: TNF-␣ regulates calcium handling in PV cardiomyocytes, which may increase PV arrhythmogenic activity and underlie the genesis of inflammation-induced atrial fibrillation. P4-4 EFFECTS OF INCREASING ICA ON SPIRAL WAVE DYNAMICS: RESULTS FROM A 2-D EXPERIMENTAL MODEL Marvin Chang, BS, Yibing Zhang, PhD, Roland Emokpae, Jr., BS, Eduardo Marba´n, MD, PhD, Leslie Tung, PhD and M. Roselle Abraham, MD. Johns Hopkins University, Baltimore, MD. Abnormal Ca2⫹ handling resulting in triggered activity underlies some ventricular tachycardias. Numerical studies show that with progressive increase of Ca current (ICa) amplitude, spiral wave reentry changes from a stable, periodic phenotype to increasing degrees of meander, culminating in spiral wave breakup. However, there is a paucity of experimental data examining the effect of increased ICa on spiral wave dynamics. Aim: We investigated the effect of (-)BayK 8644, a selective dihydropyridine receptor agonist on spiral wave dynamics. Methods: Human mesenchymal stem cells (MSC) and neonatal rat ventricular myocytes (NRVM) were coplated and optically mapped after 8-10

S216 days in culture using di-4-ANEPPS. After initiation of stable reentry by rapid pacing, cells were perfused with 2.5uM BayK for 10 min. Results: BayK produced triggered activity and increased reentry cycle length (CL) by 22% (p⬍0.01, n ⫽7), but had no effect on CL variability. Additionally, APD80 during reentry was increased by 17% (p⬍0.01, n⫽7). Tracking of reentry tip trajectories revealed decreased path length and no spiral wave breakup after BayK. An example of tip trajectories before (A) and after addition of BayK (B) is shown. Conclusions: In MSC-NRVM co-cultures, increasing ICa did not increase tip meandering or induce breakup of spiral waves. In this system, the electrically coupled but inexcitable MSCs may increase the electrical load on NRVMs with a resultant stabilizing effect. Also, increased ICa would increase Ca-dependent inactivation of ICaL, limiting APD prolongation and de-stabilization of spiral waves.

Heart Rhythm, Vol 2, No 5, May Supplement 2005 response originates at two locations (Fig. D), similar to anodal stimulation in resting tissue. These excitation patterns correspond to break excitation. For systolic stimulation, the delay of Cai response after S2 onset was about 30 ms. Conclusion: Cai transients exhibit make and break excitation modes for unipolar cathodal and anodal stimulation.

P4-6 PATTERNS OF WAVE BREAK LOCATIONS IN VENTRICULAR FIBRILLATION Bum-Rak Choi, PhD, Woncheol Jang, PhD and Guy Salama, PhD. University of Pittsburgh, Pittsburgh, PA and Duke University, Durham, NC.

P4-5 OPTICAL MAPPING OF CALCIUM DISTRIBUTION REVEALS MAKE AND BREAK EXCITATION MODES Veniamin Y. Sidorov, PhD, Marcella C. Woods, BS, Mark R. Holcomb, MS and John P. Wikswo, PhD. Vanderbilt University, Nashville, TN. Background: It is known that electrical stimulation of cardiac tissue can occur by make or break mechanisms and that virtual electrode polarization underlies these phenomena. Additionally, calcium transients can play a key role in slow conduction and induction of reentry. In this study we investigated the intracellular calcium (Cai) distribution during stimulation in systolic and diastolic cardiac tissue. Methods: We used optical imaging to measure Cai transients in 5 rabbit hearts stained with the calcium-sensitive dye Rhod-2 AM. The fluorescence was excited by a 532 nm laser, and the emitted light was filtered (585⫾20 nm) and collected by a CCD camera with spatial and temporal resolution of 128x128 pixels and 2 ms/frame. Cathodal and anodal stimuli of 20 ms duration and magnitudes of 3 and 5 mA were tested. Results: For diastolic stimulation the Cai distribution patterns were similar to previously well-described images of virtual cathode polarization. The Cai response initiated at two areas oriented along the fiber direction when positive (anodal) current was applied (Fig. A), and at the central region when negative (cathodal) current was delivered (Fig. C). This behavior corresponds to the make excitation mode of virtual electrode stimulation. The delay of Cai response after S2 onset was approximately 10 ms for diastolic stimulation. During anodal stimulation in systole (150 ms S1-S2 coupling interval), the Cai wave spreads from the image center (Fig. B), resembling diastolic cathodal stimulation. If stimulation is negative the Cai

Ventricular fibrillation (VF) has been characterized as disordered contractions of the heart with complex EKG patterns due to irregular electrical wave propagation. A better understanding of wave propagation in VF is important to elucidate its initiation, maintenance. We investigated patterns of wave propagation in VF by detecting the sites where wave fronts collide or split and correlating these locations with epicardial characteristics at those sites such as anatomical features (i.e. coronary vessels), fiber orientation and local refractoriness. Wave fronts were tracked by measuring voltage (Vm) oscillations fluorometrically from multiple locations using the voltage sensitive dye, di-4 ANEPPS and a 100x100 pixel CMOS camera (SciMedia) at 1000 frames/s from 1x1 cm2 of the anterior surface of perfused guinea pig hearts (n⫽4), followed by burst pacing to induce VF. Propagation wave fronts were automatically analyzed using a region based image analysis algorithm from at least 8 s of VF. In VF, waves split into multiple daughter wavelets at a frequency of 34⫾4 splits/s in the field of view; wavebreaks occurred for various reasons: a) decremental conduction (49⫾7%), b) collisions of several wave fronts (32⫾8%), c) increased refractoriness of the previous beats (17⫾2%). There was no statistically significant relationship between wavebreak locations and coronary vessels. Wave propagation preceding a wavebreak was mostly parallel to the transverse axis of the fibers for wavebreaks elicited by an abrupt change in refractoriness (dominant angle ⫽ 17 ⫾ 8o). In conclusion, wave breaks did not show statistically significant preferential locations in the anterior region of hearts such as vessels but were due to dynamic properties of the myocardium (i.e. restitution) and cell-cell coupling along fiber orientations. P4-7 SPATIAL HETEROGENEITY OF ACTION POTENTIAL DURATION RESTITUTION IN HUMANS Martyn P. Nash, PhD, Chris P. Bradley, PhD, Peter M. Sutton, PhD, Martin Hayward, MD, FRCS, David J. Paterson, PhD and Peter Taggart, MD, PhD. Bioengineering Institute, University of Auckland, New Zealand, University of Oxford, Oxford, United Kingdom and University College London Hospitals, London, United Kingdom.