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Oxidative phosphorylation in myocardial mitochondria 'in situ': a calorimetric study on permeabilized cardiac muscle preparations

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Abstract

A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100–500 µg were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37°C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 µM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 µM at ractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 µM α-cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H+ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 ± 3.5 mW cm-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells. (Mol Cell Biochem 174: 101–113, 1997)

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Authors and Affiliations

  1. Institut für Normale und Pathologische Physiologie, Universität Marburg, Marburg, Germany

    Detlef Köhnke & Marion Schramm

  2. Institut für Normale und Pathologische Physiologie, Universität Marburg, Marburg, Germany

    Jürgen Daut

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  1. Detlef Köhnke
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  2. Jürgen Daut
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  3. Marion Schramm
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Köhnke, D., Daut, J. & Schramm, M. Oxidative phosphorylation in myocardial mitochondria 'in situ': a calorimetric study on permeabilized cardiac muscle preparations. Mol Cell Biochem 174, 101–113 (1997). https://doi.org/10.1023/A:1006838509090

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