David A. Harris

Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion.

Heart tissue is remarkably sensitive to oxygen deprivation. Although heart cells, like those of most tissues, rapidly adapt to anoxic conditions, relatively short periods of ischaemia and subsequent reperfusion lead to extensive tissue death during cardiac infarction. Heart tissue is not readily regenerated, and permanent heart damage is the result. Although mitochondria maintain normal heart function by providing virtually all of the hearts ATP, they are also implicated in the development of ischaemic damage. While mitochondria do provide some mechanisms that protect against ischaemic damage (such as an endogenous inhibitor of the F1Fo-ATPase and antioxidant enzymes), they also possess a range of elements that exacerbate it, including ROS (reactive oxygen species) generators, the mitochondrial permeability transition pore, and their ability to release apoptotic factors. This review considers the process of ischaemic damage from a mitochondrial viewpoint. It considers ischaemic changes in the inner membrane complexes I-V, and how this might affect formation of ROS and high-energy phosphate production/degradation. We discuss the contribution of various mitochondrial cation channels to ionic imbalances which seem to be a major cause of reperfusion injury. The different roles of the H+, Ca2+ and the various K+ channel transporters are considered, particularly the K+(ATP) (ATP-dependent K+) channels. A possible role for the mitochondrial permeability transition pore in ischaemic damage is assessed. Finally, we summarize the metabolic and pharmacological interventions that have been used to alleviate the effects of ischaemic injury, highlighting the value of these or related interventions in possible therapeutics.

Dimerization of F0F1ATP synthase from bovine heart is independent from the binding of the inhibitor protein IF1.

Solubilization of heavy bovine heart mitochondria with Triton X-100 leads to the selective extraction of F0F1ATP synthase monomer and dimer in a 2:1 ratio, as revealed by blue native gel electrophoresis (BN-PAGE). Second dimensional SDS-PAGE and immunoblotting with IF1 and F1 antibodies following BN-PAGE show that both aggregation states of the ATP synthase contain IF1. The monomer/dimer ratio does not change in extracts from mitochondria subjected to different energy conditions accompanied by IF1 binding modulation or from submitochondrial particles differing in IF1 content. In addition, the usual monomer/dimer ratio is observed even in submitochondrial particles deprived of IF1. Histochemical staining for ATPase activity demonstrates that the dimer is inactive, irrespective of its IF1 content. It is concluded that in the membrane of bovine heart mitochondria the ATP synthase dimer is a stable inactive structure, whose formation is not mediated by IF1 binding.

The binding and release of the inhibitor protein are governed independently by ATP and membrane potential in ox-heart submitochondrial vesicles.

(1) The effects of membrane potential (delta psi) and nucleotides on the interaction between the F1-ATP synthase and its natural inhibitor protein (IF1) are studied in ox-heart submitochondrial vesicles. (2) Membrane potential causes displacement of IF1 from submitochondrial vesicles, as shown by measuring both delta psi-dependent stimulation of ATPase capacity and release of 125I-labelled IF1 from the vesicles. These effects are abolished if ATP is included in the incubation. (3) There is a linear increase in the steady-state ATPase capacity of oxidising vesicles as delta psi is increased from 100 mV to 135 mV. Increasing delta psi above 140 mV leads to no further change. (4) At a constant membrane potential, ATP suppresses the increase in ATPase capacity, with a concentration for half maximal effect of 140 microM. This value is close to the Km for ATP hydrolysis by membrane-bound F1. This suppression is related to ATP concentration rather than to delta Gp or ATP/ADP ratio. (5) The unidirectional on- and off-rates of IF1 were measured separately. The off-rate of IF1 is increased by membrane potential but unaffected by ATP. The on-rate, conversely, is increased by ATP. Thus, the suppression of the potential-dependent net release of IF1 from submitochondrial vesicles by ATP results from an increase of the IF1 on-rate above the off-rate.

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.

The mitochondrial ATP synthase inhibitor protein binds near the C-terminus of the F1 β-subunit

The specific, mitochondrial ATP synthase protein (IF1) was covalently cross‐linked to its binding site on the catalytic sector of the enzyme (F1‐ATPase). The cross‐linked complex was selectively cleaved, leaving IF1 intact to facilitate the subsequent purification of the F1 fragment to which IF1 was cross‐linked. This fragment was identified by sequence analysis as comprising residues 394–459 on the F1 β‐subunit, near the C‐terminus. This finding is discussed in the light of secondary structure predictions for both IF1 and the F1 β‐subunit, and sequence homologies between mitochondrial and other ATP synthases.

Azide as a probe of co-operative interactions in the mitochondrial F1-ATPase

(1) The hydrolytic activity of the isolated mitochondrial ATPase (F1) is strongly inhibited by azide. However, at very low ATP concentration (1 microM or less), no inhibition by azide is observed. (2) The azide-insensitive ATPase activity represents a high-affinity, low-capacity mode of turnover of F1. This is identified with the low Km, low Vmax component seen in steady-state kinetic studies in the absence of azide. (3) The azide-insensitive ATPase activity shows simple Michaelis-Menten kinetics, with Km = 3.2 microM, and Vmax = 1.1 mumol/min per mg (6 s-1). It is unaffected by anions such as sulphite, or by increasing pH in the range 7 to 8, both of which stimulate the maximal activity of F1. (4) Both the azide-insensitive and azide-sensitive components of F1-ATPase activity are equally inhibited by labelling the enzyme with 7-chloro-4-nitrobenzofurazan, by binding the natural inhibitor protein, or by cold denaturation of the enzyme. (5) It is concluded that azide-insensitive ATP hydrolysis represents catalysis by F1 involving a single catalytic site, and that azide acts by abolishing intersubunit cooperativity between the three catalytic sites of F1. Azide-sensitivity is thus a useful probe for events which affect the active site of F1 directly.

Reversible modulation of the mitochondrial ATP synthase with energy demand in cultured rat cardiomyocytes

Mitochondria; Cardiomyocyte; ATP synthase; ATPase inhibitor protein; Metabolic regulation; Anoxia

Control of mitochondrial ATP synthase in rat cardiomyocytes: effects of thyroid hormone.

Activities of the mitochondrial ATP synthase and the electron transfer chain were investigated in cultured cardiomyocytes prepared from untreated and thyroxine-treated rats. Quiescent cells from the thyroxine-treated animals showed a 33% increase in mitochondrial ATP synthase capacity, but no change in respiratory chain capacity, relative to those from control animals. This increase was attributable largely to (a) a 25% increase in F1 content in these mitochondria, and partly to (b) a 10% stimulation in ATPase activity due to raised intramitochondrial Ca2+. Both types of cell showed a normal ATP content of 38-40 nmol/mg cell protein. In control cells, the mitochondrial ATP synthase responded to increased energy demand (by electrical stimulation and/or by positive inotropic agents) with an increase in its capacity of up to 2-fold. This response was absent in cells from thyroxine-treated animals. In addition, cellular ATP levels fell significantly after 2 min electrical stimulation of cells from thyroxine-treated animals, while those of control cells were constant. It was concluded that regulation of the mitochondrial ATP synthase was defective in heart cells from thyroxine treated rats, leading to an energy deficit when energy demand on the cells was increased. Animals treated with thyroxine, but allowed to recover for 17 days after treatment, showed responses indistinguishable from the control cells. Thus, the effects of thyroxine on mitochondrial activities were reversible.

The ectopic FOF1 ATP synthase of rat liver is modulated in acute cholestasis by the inhibitor protein IF1

Rat liver plasma membranes contain FOF1 complexes (ecto-FOF1) displaying a similar molecular weight to the mitochondrial FOF1 ATP synthase, as evidenced by Blue Native PAGE. Their ATPase activity was stably reduced in short-term extra-hepatic cholestasis. Immunoblotting and immunoprecipitation analyses demonstrated that the reduction in activity was not due to a decreased expression of ecto-FOF1 complexes, but to an increased level of an inhibitory protein, ecto-IF1, bound to ecto-FOF1. Since cholestasis down regulates the hepatic uptake of HDL-cholesterol, and ecto-FOF1 has been shown to mediate SR-BI-independent hepatic uptake of HDL-cholesterol, these findings provide support to the hypothesis that ecto-FOF1 contributes to the fine control of reverse cholesterol transport, in parallel with SR-BI. No activity change of the mitochondrial FOF1 ATP synthase (m-FOF1), or any variation of its association with m-IF1 was observed in cholestasis, indicating that ecto-IF1 expression level is modulated independently from that of ecto-FOF1, m-IF1 and m-FOF1.

Regulation of the mitochondrial ATP synthase is defective in rat heart during alcohol-induced cardiomyopathy

Mitochondrial ATP synthase capacity was measured in cardiomyocytes from sucrose-fed (control) and alcohol-fed (cardiomyopathic) rats. Cells from alcohol-fed rats showed an ATP synthase capacity raised by 40% relative to the control values of 1.8 mumol/min per mg cell protein. Cells from control rats were able to increase their ATP synthase capacity by up to 80% in response to increased energy demand. In contrast, cells from alcohol-fed rats had lost the ability to up-regulate their ATP synthase. Cells from control rats maintained their internal ATP levels at 38 nmol/mg cell protein before and after an increased energy demand. In cells from alcohol-fed rats, ATP levels fell by 20% after 2 min of increased energy demand. It was concluded that the inability of cells from alcohol-fed rats to maintain ATP levels was due to their reduced ability to increase mitochondrial ATP synthase activity as energy demand is increased. Such ATP deficits may contribute to heart dysfunction in alcohol-induced cardiomyopathy.