Ara Arutunyan

Locations of ectopic beats coincide with spatial gradients of NADH in a regional model of low-flow reperfusion

We studied the origins of ectopic beats during low-flow reperfusion after acute regional ischemia in excised rat hearts. The left anterior descending coronary artery was cannulated. Perfusate was delivered to the cannula using an high-performance liquid chromatography pump. This provided not only precise control of flow rate but also avoided mechanical artifacts associated with vessel occlusion and deocclusion. Optical mapping of epicardial transmembrane potential served to identify activation wavefronts. Imaging of NADH fluorescence was used to quantify local ischemia. Our experiments suggest that low-flow reperfusion of ischemic myocardium leads to a highly heterogeneous ischemic substrate and that the degree of ischemia between adjacent patches of tissue changes in time. In contrast to transient ectopic activity observed during full-flow reperfusion, persistent ectopic arrhythmias were observed during low-flow reperfusion. The origins of ectopic beats were traceable to areas of high spatial gradients of changes in NADH fluorescence caused by low-flow reperfusion.

Multiple injury approach and its use for toxicity studies.

We present an experimental approach that allows exposure of cells plated on a single coverslip to multiple distinct environments. The original chamber design created a small region of injury using geometrically defined flows of the control and ischemic solutions. Modifications of the original chamber design presented in this article produce a range of flow patterns that can be advantageous for a variety of imaging applications. These applications include: experiments that address effects of different treatments applied to a cell network, parallel testing of negative and positive controls using a single coverslip, border effect studies, evaluation of the treatment’s reversibility, and simultaneous monitoring of a cell layer loaded with different fluorescent indicators. The method also can be used to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in a normal or altered cardia cell network. These possibilities are illustrated in cultures of neonatal rat cardiomyocytes using oxidant-and calcium-sensitive fluorescent indicators.

Ischemic Boundary as a Source of Scroll Waves: A Three-Dimensional View

This study focuses on abnormal waves of activity formed within the border zone of an ischemic region. We considered three-dimensional block of cardiac tissue in which a steep gradient in cell-to-cell coupling constituted border-like conditions. This border was then placed under time-dependent conditions that promote cell automaticity and moved in space. The events mimicked complex clinical conditions associated with ischemia-reperfusion. Network behaviour was modeled based on Beeler-Reuter formalism of cardiac cell taking into account individual cell heterogeneity. The observed events ranged from single cell activity, to spherically spreading sources and multiple scroll waves. A bell-shaped relationship between the speed at which the coupling gradient moves in space and the probability of a scroll wave to escape was established. The data provide insights into possible mechanisms of ectopic activity formation and its escape from the boundary of ischemic tissue.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Method for In-Vitro Studies of Cellular Interactions at the Interface of Two Tissues

We describe a simple and reliable experimental technique that enables one to create a high fidelity linear interface between two opposing cell layers. The method employs a custom designed lid that fits a standard 3cm cell culture dish. During cell plating, the dish is divided by a 200 micron thick separator that is part of the lid. The separator is covered in a thin layer of parafilm that forms a hermetic seal with the underlying coverslip and creates a temporary gap between the two cell plating environments. After cells attach, the custom lid is replaced with a standard lid and cells are allowed to grow under standard cell culture conditions. When expanding cell layers fill the gap, a linear interface is formed between the two opposing fields. Paracrinal factors released from an approaching cell front as well as direct physical and molecular interactions between two cell types affect intercellular orientation, individual cell morphology, and the degree of cells invasion into the opposing layer. The local interface appearance thus depends on a specific cell pair and may vary dramatically. We describe several types of such interfaces for different cell pairs, including cardiomyocytes, fibroblasts, melanocytes, endothelial cells and colon carcinoma cell lines. The method serves as a practical in vitro tool to study cell growth and invasion that occur on the interface of two neighboring tissues.