Ischaemia/anoxia-induced increases in cardiac mitochondrial respiration: changes in Complex I

Abstract

Previous studies have shown that ischaemia and hypoxia induced a loss of Complex I (NADHubiquinone reductase) activity in cardiac mitochondria [l-31. We have confirmed the loss of Complex I activity after 60 min of ischaemia (with and without reperfusion), but we also reported that, by contrast, 60 min of anoxia resulted in a small increase in respiratory capacity [4]. Other groups have reported increased mitochondrial respiration after 10 rnin of hypoxia [5] and 30 min of hypoperfusion [6], the latter postulating a specific effect on Complex I. This report describes our studies on respiration and Complex I activity after short periods of ischaemia and anoxia.Male Wistar rats (200-25Og) were anaesthetised by i.p. Nembutal and the hearts were perfused in a Langendorff apparatus with a modified Krebs-Henseleit buffer containing l l m M glucose, continually gassed with either 95 % 0 2 / 5 % C02 (normoxic) or 95 % N2/5 % C 0 2 (anoxic). After 15 rnin of normoxic perfusion control hearts were perfused for a further 15-30 min in normoxic conditions, or global ischaemia was induced for 15 min by clamping off the perfusate supply, or hearts were perfused for 30 min with anoxic buffer. The ventricles were separated from the hearts and mitochondrial fractions prepared by a Nagarse digestion/ Potter homogenisation procedure. Oxygen uptake was measured in a Clark-type electrode in intact mitochondria with 5 mM glutamate/5 mM malate or 10 mM succinate (+ 2 Fg/ml rotenone) as substrates with 0.25 mM ADP, and in broken mitochondria with 250 pM NADH after three freeze-thawing cycles.The effects of ischaemia and anoxia on the respiration rates are shown in Table 1. Both ischaemia and anoxia increased glutamate/malate oxidation, by about 45%. Furthermore, these differences persisted when the mitochondria were lysed by freeze-thawing to assay NADH oxidation, which was increased by 36 % and 41 % in mitochondria from ischaemic and anoxic hearts respectively (Table 1). In contrast, the succinate rate was not different in mitochondria from ischaemic hearts. The oxidation rates were also assayed spectrophotometrically in intact mitochondria from control and anoxic hearts as the antimycin-sensitive reduction of ferricyanide [71. In this system, glutamate/malate oxidation was also increased after anoxia (689 * 25 vs. 462 f 36 nmoles FeCN.min-1

Effects of Hypo- and Hyperthyroidism on the Complex I activity in rat heart mitochondria

“…The effect on glutamate/malate may be due to the opposing effects of the thyroid state on the Complex I activity as shown above. It is interesting to note that we have previously demonstrated a similar decrease in the apparent Km for ubiquinone to explain the initial increase in NADH-dependent respiration associated with short periods of cardiac ischaemia [8].…”