The central focus of our research is on the electrophysiological properties of engineered networks of heart cells, as they relate to mechanisms underlying electrical stimulation and reentrant arrhythmias. We utilize microfabrication techniques, tissue culture, optical mapping, theoretical analysis, and computational models to study the experimental and theoretical behavior of cardiac cell networks. Our work is currently directed along the following areas: Engineered Networks of Cardiac Cells Although a great deal is known about the electrophysiology of cardiac cells at the single cell and molecular levels, their integrated behavior as organized networks in tissue is less well understood. Whereas computer models have been used to largely fill this void, an engineered, in vitro experimental model would have great value. Towards this goal, we are utilizing optical mapping to study the functional behavior of cultured monolayers of neonatal rat cardiac cells containing up to several hundred thousand cells. Photolithographic, microprinting, and microabrasion techniques are employed to cast predefined patterns of extracellular matrix proteins or topological features onto cover slips, that serve to guide the growth and spread of the cultured cells. One example of this approach is illustrated in the three photomicrographs on the left, taken at increasing magnification. The cultured cardiac cells have been grown as a hairpin loop. By using a multichannel optical mapping system developed in our lab, the electrophysiological responses of this cellular network to applied electric fields can be recorded and analyzed for comparison with theoretical and computational models. Such studies can lend insight into the role that tissue structure plays in the functional response to field stimulation. A second example of this approach is illustrated in the photomicrographs on the right. Conventional monolayers of cardiac cells contain cells that are randomly oriented. Here, we illustrate a novel method to form confluent monolayers with controlled macroscopic alignment (anisotropy), that reflects a more realistic structure that is key for many functional properties of cardiac tissue. Cells were cultured on plastic cover slips that were microabraded by lapping paper, with a cross-section shown in the scanning electron micrograph (panel A; bar is 10 microns). The architecture of the elongated and coaligned cells is shown by green staining for actin (panel B), red staining for sarcomeric a-actinin (panel C), and red staining for connexin-43 (panel D). Cell nuclei are shown in blue. Bars in panels B-D are 25 microns. Reentrant Activity of Cardiac Cell Monolayers Using a method we call "contact fluorescence imaging," the spatial propagation of excitatory wavefronts can be imaged in confluent monolayers of cultured cardiac cells. A major form of cardiac arrhythmia, reentry, can be electrically induced in these monolayers and tracked using CFI. The application of an electric field pulse can terminate the reentrant activity, and is a small-scale form of cardioversion. Electrical pulses can also accelerate the reentry by introducing additional wavefronts into the reentrant circuit. An example of a stable single loop reentry is shown below. One full rotation of the reentry is illustrated. Color bar corresponds to normalized voltage level, with blue being resting state (0%) and red being peak of the action potential (100%). Frames read from left to right, and time between frames is 20 ms. Contribution of tissue heterogeneity to arrhythmia Disease states such as ischemia can give rise to functional heterogeneities in cardiac tissue, resulting in nonuniform wave propagation that can lead to arrhythmia. To better understand the role of tissue heterogeneity in arrhythmia, many researchers have attempted to create experimental preparations with nonuniform properties. It has been a challenge, however, to achieve heterogeneities that are both highly controlled and reproducible. We have developed a novel flow chamber that allows us to superfuse two distinct areas on a cell monolayer with different solutions. This system allows us to produce a variety of electrophysiological effects in an isolated central region. The current focus of this project is to investigate how these localized heterogeneities may facilitate the formation of arrhythmias. Mechanoelectrical Feedback in the Heart Previous work in the literature on cells and tissue have shown that mechanical stretch can alter action potential duration and generate extrasystoles. It has been speculated that this process of "mechanoelectric feedback" could alter the normal distribution of repolarization and excitability, and potentiate the likelihood for reentrant arrhythmia in failing hearts. However, while this hypothesis is attractive, experimental studies that demonstrate stretch-induced reentry are few and indirect. In this aspect of our work, we use a miniature nozzle to jet a tiny pulse of solution against the surface of a cultured monolayer of cardiac cells. This form of mechanical stimulus can induce a wavefront of electrical activity from the point of impingement. Shown in the figure on the right, left panel, is an activation wave spreading from a point electrode. In the right panel is a similar wave of activation, but resulting from the mechanical pulse. Experiments are underway to characterize the how mechanical stretch, pressure and shear stress may alter the electrophysiological properties of tissue systems. The ultimate goal is to determine those mechanical conditions that may facilitate the initiation of arrhythmic activity. Cardiac Tissue Engineering We are working to synthesize thin layers of three-dimensional, engineered cardiac tissue. One approach is to mix isolated cardiac cells or embryonic stem cells into gels and to culture them in tissue culture plates. The expression of cardiac-specific genes and proteins can then be characterized in these tissue constructs, followed by functional electrophysiological and mechanical assays. We are also introducing foreign genes to overexpress subunits of certain ion channels, thereby altering the electrophysiological characteristics of the cultured cells.

Maintained by: Les Tung
Last Updated On: 12/9/06