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Am J Physiol Heart Circ Physiol 274: H2001-H2008, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 6, H2001-H2008, June 1998

A superfusion system to study border zones in confluent cultures of neonatal rat heart cells

Christopher J. Hyatt1, John J. Lemasters3, Barbara J. Muller-Borer2, Timothy A. Johnson1, and Wayne E. Cascio2

1 Department of Biomedical Engineering and Division of Cardiology, 2 Department of Medicine, and 3 Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220-320 µm wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.

intracellular pH; gap junctions; ischemia; arrhythmia; monolayer


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