Subcellular distribution of endogenous long chain acylcarnitines during hypoxia in adult canine myocytes

Abstract

Previous studies from our laboratory showed a pronounced increase in the sarcolemmal accumulation of long chain acylcarnitines in isolated neonatal rat myocytes after a prolonged (60 minute) hypoxic interval. Because much shorter intervals of hypoxia are associated with electrophysiological alterations in adult cells, the present study was performed to assess the extent of sarcolemmal accumulation of long chain acylcarnitines during hypoxia in adult canine myocytes. Cells were incubated (for 24 hours) with 3H-carnitine and the uniformity of incorporation into carnitine fractions was verified biochemically. Cells were exposed to hypoxia (PO2 < 15 mm Hg) for 10 or 20 minutes in the presence or absence of sodium 2-[5-(4-chlorphenyl)-pentyl]-oxirane-2-carboxylate (POCA; 10 microM), an inhibitor of carnitine acyltransferase I. Cells were processed for electron microscopical autoradiography with a technique to spatially fix endogenous long chain acylcarnitines with selective and complete removal of short chain and free carnitine. Grain distributions were analysed by the maximum likelihood method from digitised micrographs. Total mass of long chain acylcarnitines increased ninefold [42.3(3.3) to 374(42) pmol.mg-1 protein] by 10 minutes of hypoxia and 15-fold [to 632(36) nmol.mg-1 protein] by 20 minutes of hypoxia. Normoxic cells exhibited little long chain acylcarnitines in the sarcolemma, and modest amounts in mitochondria and cytoplasm. In hypoxic cells, content of long chain acylcarnitines in mitochondria and cytoplasm increased by a maximum of twofold. By contrast, long chain acylcarnitines increased 100-fold in the sarcolemma to 4.18 x 10(6) molecules.microns-3 after 10 minutes of hypoxia. The increase in long chain acylcarnitines with hypoxia was completely prevented by pretreatment with POCA. Hypoxia in adult ventricular myocytes induces a rapid and preferential increase in endogenous long chain acylcarnitines within the sarcolemma.