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(Circulation Research. 2006;99:165.)
© 2006 American Heart Association, Inc.

Cellular Biology

Dynamic Responses of Single Cardiomyocytes to Graded Ischemia Studied by Oxygen Clamp in On-Chip Picochambers

Vladimir Ganitkevich, Sibylle Reil, Brigitta Schwethelm, Thomas Schroeter, Klaus Benndorf

From the Institut für Physiologie II (V.G., S.R., K.B.), Friedrich-Schiller-Universität; and Institut für Fügetechnik und Werkstoffprüfung GmbH (B.S., T.S.), Jena, Germany.

Correspondence to Dr K. Benndorf, Friedrich-Schiller-Universität Jena, Institut für Physiologie II, D-07743 Jena, Germany. E-mail Klaus.Benndorf{at}mti.uni-jena.de

Single mouse cardiomyocytes were exposed to defined ischemia. We designed chambers on glass chips with a volume of 192 pL (picochambers). After a picochamber was loaded with a single cardiomyocyte, PO₂ in the picochamber was equilibrated with that in the headspace, where it was controlled in the critical range between <0.2 and 10 mm Hg. Because the extracellular fluid volume in a picochamber was restricted, these conditions are close to tissue ischemia. Responses of the sarcolemmal K_ATP-channel current (I_KATP), the production of reactive oxygen species (ROS), and the mitochondrial membrane potential () of single cardiomyocytes to graded ischemia and, in particular, to rapid changes of the ischemic grade by defined oxygen steps were studied. The results show that I_KATP is readily activated during ischemia and that the grade of ischemia tightly controls the amplitude of I_KATP. Furthermore, maximal ischemia-induced I_KATP was similar when it followed either reoxygenation or reperfusion, suggesting that there is no major autocrine modulation of maximal I_KATP during ischemia. A PO₂ staircase from <0.2 to 10 mm Hg increased the ROS signal, starting already at a PO₂ of 0.3 mm Hg. With a similar PO₂ staircase, first hyperpolarized and then, above 1 mm Hg, depolarized. The depolarizing response of at a PO₂ of >1 mm Hg could be blocked by increasing the antioxidant defense with glutathione–monoethyl ester. It is concluded that in an ischemic cardiomyocyte I_KATP is essentially controlled by PO₂ and that at low PO₂ is balanced by oxygen-induced hyperpolarization and ROS-induced depolarization.

Key Words: single cardiomyocytes • ischemia • reactive oxygen species • mitochondrial membrane potential • ATP-sensitive K⁺ channels

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