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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, P(O2) in the picochamber was equilibrated with that in the headspace, where it was controlled in the critical range between &lt;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 (delta psi) 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 P(O2) staircase from &lt;0.2 to 10 mm Hg increased the ROS signal, starting already at a P(O2) of approximately 0.3 mm Hg. With a similar P(O2) staircase, delta psi first hyperpolarized and then, above 1 mm Hg, depolarized. The depolarizing response of delta psi at a P(O2) of &gt;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 Po(2) and that at low P(O2) delta psi is balanced by oxygen-induced hyperpolarization and ROS-induced depolarization.

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