Inhibition of mitochondrial β-oxidation in the heart by increased redox state of the ubiquinone pool

Abstract

Mitochondria1 P-oxidation is linked to the respiratory chain at two stages; 3-hydroxyacyl-CoA dehydrogenase to the ubiquinone pool via NAD and complex I [I], and acyl-CoA dehydrogenases to the ubiquinone pool via electron transfer flavoprotein (ETF) and ETF:ubiquinone oxidoreductase [2]. Inhibition of the respiratory chain, as occurs in cardiac ischaemia, could therefore lead to inhibition of P-oxidation either by inhibition of 3-hydroxyacyl-CoA dehydrogenase or the acyl-CoA dehydrogenases. Studies carried out in the perfused heart observed accumulation of 3-hydroxyacyl-esters during ischaemia, suggesting inhibition of the 3-hydroxyacyl-CoA dehydrogenase [3,4]. However, other studies found that 3-hydroxyacyland 2-enoyl-CoA esters only formed a small percentage of the CoA esters accumulated [S] and that incubation of intact mitochondria with cyanide, mimicking cardiac ischaemia, did not lead to accumulation of 3-hydroxyacyl-esters but incubation with rotenone, a complex I inhibitor, did lead to their accumulation [6].In order to resolve the question of which step of P-oxidation is inhibited during cardiac ischaemia, we incubated cardiac mitochondria with 90pM [U-'4C]hexadecanoyl-CoA and measured P-oxidation flux and intact CoA esters [7] accumulated during inhibition of the respiratory chain with increasing amounts of myxothiazol, a complex 111 inhibitor. Parallel incubations were used to measure the redox states of the NAD'MADH pool and ubiquinondubiquinol pools [8,9]."C02 release and production of acid soluble radioactivity and acetyl-carnitine were progressively inhibited by myxothiazol ( Figure 1 ). The mAD']/pADH] ratio during 0-oxidation of hexadecanoyl-CoA decreased from 17 in the absence of myxothiaxol to 3 in the presence of 20pM myxothiazol. Similarly, the ratio [ubiquinone]/[ubiquinol] decreased from 9 in the absence to 0.4 in the presence of 20pM myxothiazol. During inhibition of P-oxidation by myxothiazol, the amounts of 3-hydroxyhexadecanoyl-CoA and 2hexadecenoyl-CoA increase relative to their chain shortened intermediate, tetradecanoyl-CoA (Figure 2). The presence of consistent amounts of 3-hydroxyhexadecanoyl-CoA and 2hexadecenoyl-CoA during inhibition of flux by 75% suggests that inhibition of the 3-hydroxyacyl-CoA dehydrogenase is involved. However, 3-hydroxyacyl-and 2-enoyCCoA esters do not increase to the same extent as in succinate-induced reverse electron flow [ 101 or 3-hydroxyacyl-CoA dehydrogenase deficiency [ 1 1 ] so that inhibition of the acyl-CoA dehydrogenases must be involved as well. A Y o 2 release v acid soluble product 0 acetyl-carnitine + total "C products 20 0 0.2 0.4 0.6 20 [myxothiazoll IpM) Figure 1 Products of mitochondrial P-oxidation of [U-''C]hexadecanoyl-CoA during inhibition by myxothiazol. Meanf SEM for at least 3 measurements I nn relcnlmn l i m e ( m m ) Figure 2 Radio-HPLC chromatogram showing accumulation of acyl-CoA esters generated from [U-'4C]hexadecanoyl-CoA by rat heart mitochondria during inhibition of P-oxidation. [A] no myxothiazol, [B] 0.2pM myxothiazol...