optical mapping of anatomical reentry - Biomedical Engineering

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During experiments, the cells were stained with the voltage- sensitive dye di-8-ANEPPS (70μM, 5min), and superfused with normal Tyrode's solution at 34-37 oC ...
OPTICAL MAPPING OF ANATOMICAL REENTRY IN MONOLAYERS OF CULTURED NEONATAL RAT CARDIAC MYOCYTES Steven N. Lu, Emilia Entcheva, Vinod Sharma, Leslie Tung Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD Email: [email protected] Abstract—There were two primary objectives in this study. First, we present a novel macroscopic optical method with high sensitivity to record transmembrane potentials, Vm, in monolayers of cultured cardiac myocytes. Second, using this technique we demonstrate, for the first time, electricallyinduced, sustained reentrant activity in a two-dimensional (2-D) cell culture preparation. Index Terms—cardiac electrophysiology, cell culture I. INTRODUCTION Reentry is the mechanism underlying a major class of cardiac arrhythmias. It has been extensively studied in excised tissue preparations and whole heart. In this paper we introduce a new, well-controlled, 2-D anatomical reentry model. Sustained reentrant activity is electrically induced in monolayers of cultured neonatal rat cardiac cells and recorded using a novel method for optical mapping (“contact fluorescent imaging”, CFI) of cardiac Vms. II. METHODS Confluent monolayers were grown on circular coverslips (22mm dia., 150µm thick), from 3-4 day old Sprague-Dawley rat cardiac myocytes, isolated using published procedures [1]. During experiments, the cells were stained with the voltagesensitive dye di-8-ANEPPS (70µM, 5min), and superfused with normal Tyrode’s solution at 34-37 οC. The excitation light (510-560nm) was delivered to the preparation via a liquid light guide from above. A custom fabricated bundle of 48 plastic optical fibers (1mm dia., NA 0.5) was positioned at the bottom of the coverslip to collect the emitted fluorescent signals (indicating Vm). The signals were filtered (λ > 590nm) and detected using photodiodes and custom designed electronics. For studying reentry, an anatomical obstacle (6 x 0.5mm) was cut in the monolayer using a syringe needle. The cells were conditioned by pacing via field stimulation (S1, 2Hz, >2min). Using the same field electrodes, a premature stimulus (S2, ~60ms S1-S2 coupling interval) was delivered after the last S1 to assist in the induction of reentry. III. RESULTS Optically recorded action potentials from these monolayer preparations at 1mm spatial resolution are free of contraction motion artifact at normal extracellular [Ca++], since cell adhesion to the glass mechanically restricts the monolayer. Four episodes of sustained anatomical reentry were induced in the monolayer preparations and recorded using CFI at 1mm spatial resolution (Fig. 1). Average conduction velocity and period during reentry were 10.8cm/s and 140ms.

Figure 1: A: A top view schematic of the glass coverslip, with the fiber bundle drawn to scale. The black circles represent the location of the obstacle, and arrows, the direction of reentry propagation. Channels 1-14 are labeled. B: Raw signal (cutoff f=500Hz) recorded from channel 1 during a reentry episode. C: Activation contours (50% APA, 9 ms apart) of the first reentry cycle shown in Panel B. D: Frame-stack (channels 1-14) for the 1 second of the reentry shown in panel B. White signifies depolarized and black - repolarized tissue.

IV. DISCUSSION The CFI method provides sufficient sensitivity to optically measure Vm from a single layer of cells. The fiber bundle can be constructed to match arbitrary geometry and field-of-view. We found that it is possible to induce sustained (several minutes) reentrant activity in these monolayer preparations. Reentry was also successfully terminated using a single higher strength electrical pulse. We are currently developing reentry models with better-controlled geometries using cell patterning techniques [2]. V. CONCLUSIONS The results reported in this study suggest the monolayer preparation to be an attractive alternative to tissue models of reentry and defibrillation. CFI can provide the means to monitor Vm in such preparations. VI. ACKNOWLEDGMENTS This work was supported by NIH grant 48266. [1] [2]

VII. REFERENCES D. C. Johns, et al., “Adenovirus-mediated expression of a voltagegated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver)”, J Clin Invest, vol. 96, pp. 1152-8, 1995. S. Rohr, D. Shoelly, and A. Kleber, “Patterned growth of neonatal rat heart cells in culture. Morphological and electrophysiological characteristics.”, Circ Res, pp. 114-130, 1991.