The effects of ischaemic preconditioning, diazoxide and 5‐hydroxydecanoate on rat heart mitochondrial volume and respiration

Abstract

Studies with different ATP-sensitive potassium (K ATP ) channel openers and blockers have implicated opening of mitochondrial K ATP (mitoK ATP ) channels in ischaemic preconditioning (IPC). It would be predicted that this should increase mitochondrial matrix volume and hence respiratory chain activity. Here we confirm this directly using mitochondria rapidly isolated from Langendorffperfused hearts. Pre-ischaemic matrix volumes for control and IPC hearts (expressed in ml per mg protein ± S.E.M., n = 6), determined with 3 H 2 O and [14 C]sucrose, were 0.67 ± 0.02 and 0.83 ± 0.04 (P < 0.01), respectively, increasing to 1.01 ± 0.05 and 1.18 ± 0.02 following 30 min ischaemia (P < 0.01) and to 1.21 ± 0.13 and 1.26 ± 0.25 after 30 min reperfusion. Rates of ADP-stimulated (State 3) and uncoupled 2-oxoglutarate and succinate oxidation increased in parallel with matrix volume until maximum rates were reached at volumes of 1.1 ml ml _1 or greater. The mitoK ATP channel opener, diazoxide (50 mM), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2-oxoglutarate oxidation. Direct addition of diazoxide (50 mM) to isolated mitochondria also inhibited State 3 succinate and 2-oxoglutarate oxidation by 30 %, but not that of palmitoyl carnitine. Unexpectedly, treatment of hearts with the mitoK ATP channel blocker 5-hydroxydecanoate (5HD) at 100 or 300 mM, also increased mitochondrial volume and inhibited respiration. In isolated mitochondria, 5HD was rapidly converted to 5HD-CoA by mitochondrial fatty acyl CoA synthetase and acted as a weak substrate or inhibitor of respiration depending on the conditions employed. These data highlight the dangers of using 5HD and diazoxide as specific modulators of mitoK ATP channels in the heart.

Ultrastructural and functional abnormalities of mitochondria in cultivated fibroblasts from α-mannosidosis patients

“…An increase in the mitochondrial matrix volume correlates well with an increase in respiration rates as was already demonstrated by several studies (Halestrap 1987(Halestrap , 1989Lim et al 2002). It could be in agreement with our observation of increased activities of respiratory chain complexes.…”

“…This decrease can consequently trigger secondary-induced adaptively-compensatory increase of cellular respiration without any changes in protein amount. Nevertheless, activation of mitochondrial respiratory chain enzymes could be associated with mitochondrial swelling as well (Halestrap 1987(Halestrap , 1989Lim et al 2002). Recently Kaasik et al (2007a) postulated that organelle transport in neuronal processes is a basis for neuronal functions.…”

“…Kaasik et al (2007b) hypothesized that during energetic stress, when the demand for ATP is high and mitochondrial potential decreases, an increase in matrix volume could further activate the respiratory chain. It is interesting to note that matrix volume is increased in ischemic mitochondria as well as following reperfusion (Lim et al 2002). In addition, mitochondrial swelling could play a role in triggering of apoptosis via volume-depedent cytochrome c releasing (Gogvadze et al 2004).…”

Mitochondrial K channels in cell survival and death

“…However, since opening of mitoK ATP causes only a small change in K + concentrations in the mitochondria, other nonspecific effects of mitoK ATP openers can mask their effects on respiration. Diazoxide has been shown to inhibit succinate-supported respiration by inhibiting succinate dehydrogenase (Complex II) without affecting NADHsupported respiration [36,37]. Thus, when NADH-linked substrates are used, mitochondrial oxygen consumption is increased by ∼20% in the heart and liver [38] and ∼50% in the brain [35,39] (due to higher levels of mitoK ATP in the brain).…”

“…Other SDH inhibitors, malonate and 3-nitropropionic acid (3-NPA), have also been shown to attenuate oxidant stress on the heart and have cardioprotective effects [62][63][64]. These observations have led to the alternative hypothesis that respiratory inhibition, rather than K + channel activation, underlies IPC [36,37]. The pharmacological overlap between SDH and mitoK ATP led to the hypothesis that SDH and mitoK ATP interact with each other, both physically and functionally [65].…”