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 can 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. Photolithography, microcontact printing, 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 photomicrographs on the right. Conventional monolayers of cardiac cells contain cells that are randomly oriented. Here, we illustrate a simple 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.
Another example of this approach is illustrated in the photomicrograph on the left. Using soft lithography and microcontact printing, the cultured cardiac cells have been grown in a zigzag pattern (a = 0.3 mm, b = 1.5 mm; width of cell strand = 0.1 mm). By using a multichannel optical mapping system developed in our lab, the propagation of electrical wavefronts around and through this patterned region can be recorded and studied in ways that were previously possible only with computational models. Such research provides insight into the role that tissue structure plays in the genesis of conduction block and formation of reentrant arrhythmia.
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 drifting 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.
Contribution of Tissue Heterogeneity to Arrhythmia
Disease states such as ischemia, border zones of infarcts, or cardiomyopathy 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 a circular central region of the cell monolayer with a different bath solution. This system allows us to produce a variety of electrophysiological changes in the central region.
In a different approach, we have used lentiviruses to overexpress or suppress targeted genes of interest with stable expression in the cardiac cells. By confining the transduced cells (shown here in green expressing eGFP) to a central region and grown in co-culture with nontransduced cells (shown here in red labeled with CMTPX), a central region of heterogeneity can again be generated, but this time with molecular rather than pharmacological perturbation. The current focus of these projects is to investigate how such localized heterogeneities may facilitate the formation of arrhythmias.
Cardiac Fibrosis
Cardiac fibrosis is a ubiquitous, pathological condition found in old or diseased hearts in which cardiac cells are separated by collagenous septa. We are working to develop an in vitro model of fibrosis that can be used to test potential gene and cell-based therapies. As a first step, cardiac monolayers treated with the cytokine TGF-beta show significantly higher levels of interstitial collagen (indicated by picrosirius red stain). We are refining this model further by controlling the orientation and spacing of the cardiac cells.
