Pacemakers, like bandleaders conducting the musicians in their group, generate rhythmic signals that control the activities of biological systems. In a human heart, the pacemaker is composed of roughly 5000 cells in the sinoatrial node, located in the right roof of the atrium. Regulated by chemical signals from the nervous system, the pacemaker cells coordinate their rhythm to produce a single impulse that is propagated to the rest of the heart during each cardiac cycle.
In efforts that may improve understanding of how natural pacemakers form and provide such steady signals, researchers at Technion University in Israel studied the development of a pacemaker in an artificial network composed of rat cells which, intriguingly, were not originally part of a pacemaker system. Although the resulting artificial network is very different from a heart, it can provide insights into how the structure and density of a biological system affect the development of pacemakers.
In their experiment, the researchers excised muscle cells (myocytes) and connective tissue cells (fibroblasts) from the rats' ventricles. They deposited the cells at the desired density on collagen-coated glass petri dishes where the cells thrived and grew in the presence of nutrients and other proper incubating conditions. The plated cells (i.e. myocytes and fibroblasts) proliferate, migrate and assemble themselves into a a flat, two-dimensional network and later on to a network of fibers, if the initial cell density is high enough.
The initial proportion of myocytes versus fibroblasts not only influences the shape and structure of the network that they form, but also affects the rhythmic patterns of cell contraction. Each cell configuration evolves differently and often results in a different shape and structure. Within 2-4 days, sufficiently dense cultures form a single layer of cells (first figure below) that becomes progressively thicker and finally evolves (within 1-3 weeks) into a network of fibers (second figure below).
Using a CCD camera (with a time resolution of 40 milliseconds) and real-time computer processing, the researchers developed a noninvasive optical recording technique for continuously following the development and behavior of the network. The technique allows a simultaneous recording of 120 days of network formation and cell movements, including patterns of contraction in the muscle cell. Recording and analyzing the latter is only limited by the capacity of the hard disk which, in the reported experiment, was sufficient for 2 weeks of continuous recording.
A special computer program was developed to acquire the incoming video frames, designating regions of interest (marked by the boxes that appear in the two images shown above), and detect the motion associated with cell contraction within each region. Detecting the cell contractions is achieved by measuring differences in brightness between subsequent video frames. By adjusting the size of the field of view, the researchers could study the behavior at different organization levels, from isolated cells to dense networks, either in the early or late stages of development.
Continuously monitoring the network revealed interesting disorders in the rhythm of cell contractions. These included, for example, transitions between irregular and regular rhythms. From these the researchers deduced that during its evolution, the network spawned one or more pacemaker regions sending electrical signals through an otherwise quiescent medium of non-pacemaker cells. Although this is not the first evidence for rhythmic activity in networks of non-pacemaker cells, it is still the first to follow continuously how a diverse population of non-pacemaker cells orgazizes itself into functional entities such as a pacemaker and a quiescent medium.
This experiment is part of an exciting research effort to study the intrinsic properties of biological networks with cells that interact strongly. This particular system is formed by taking a part of the rat heart, decomposing it into its cell constituents and letting it reorganize on a two-dimensional surface. Thus, physiologically, it is very different from the heart, and consequently, it cannot be employed to draw any conclusions regarding the development and operation of an intact heart. Nevertheless, a systematic examination of such systems can potentially reveal the relations between the density, shape, and structure of the network and its contractile characteristics, thereby shedding more light on our understanding of pacemaker formation and stability. (Images courtesy of the authors; thanks to Yoav Soen for help with the text.)
This research is reported by Yoav Soen, Netta Cohen, Doron Lipson, and Erez Braun, Physical Review Letters 82, 3556 (26 April 1999).