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Subcellular Distribution and Analysis of Technetium-99m-MIBI in Isolated Perfused Rat Hearts

Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

Correspondence: For reprints contact: David Piwnica-Worms, MD, PhD, Department of Radiology, Brigham and Women's Hospital, 75 Francis St., Boston, MA02115.

ABSTRACT

To address the apparent discrepancy between cultured cells and whole heart preparations, Langendorff-perfused rat hearts loaded with hexakis (2-methoxyisobutyl isonitrile) techneticum (I) (^99mTc-MIBI) were fractionated by a standard differential centrifugation method and fractional contents of ^99mTc-MIBI were correlated with the mitochondrial marker, malate dehydrogenase (MDH), and mitochondrial substrates. The "cytosolic" fraction nominally contained 89% ± 3% of total ^99mTc-MIBI, but also contained 91% ± 1% of total MDH activity by this method. Chromatographic analysis of activity in the "cytosolic" fraction demonstrated >95% of the agent was present as the original free cationic complex; binding to a small molecular weight cytosolic protein was not involved in localization. Addition of the mitochondrial uncoupler CCCP (5 µM) to both "mitochondrial" and "cell fragment" pellets released up to 84% ± 8% of ^99mTc-MIBI content and addition of the mitochondrial substrate succinate (10 µM) in the presence of rotenone (1 µM) enhanced ^99mTc-MIBI content by up to 139% ± 52% over the control. These correlative data from rat hearts indicate that approximately 90% of ^99mTc-MIBI activity in vivo is associated with mitochondria in an energy dependent manner as a free cationic complex, but migrates during fractionation/centrifugation.

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