Richard G. Hansford

Dependence of H2O2 formation by rat heart mitochondria on substrate availability and donor age.

We have examined the substrate specificity and inhibitor sensitivity of H2O2 formation by rat heart mitochondria. Active H2O2 production requires both a high fractional reduction of Complex I (indexed by NADH/NAD+ + NADH ratio) and a high membrane potential, ΔΨ. These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H2O2 formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn2+ to give 10 nmol/mg of mitochondrial protein enhances H2O2 production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H2O2 production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).

Physiological role of mitochondrial Ca2+ transport

A model has been proposed in which mitochondrial Ca2+ ion transport serves to regulate mitochondrial matrix free Ca2+ ([Ca2+]m), with the advantage to the animal that this allows the regulation of pyruvate dehydrogenase and the tricarboxylate cycle in response to energy demand. This article examines recent evidence for dehydrogenase activation and for increases in [Ca2+]m in response to increased tissue energy demands, especially in cardiac myocytes and in heart. It critiques recent results on beat-to-beat variation in [Ca2+]m in cardiac muscle and also briefly surveys the impact of mitochondrial Ca2− transport on transient changes in cytosolic free Ca2+ in excitable tissues. Finally, it proposes that a failure to elevate [Ca2+]m sufficiently in response to work load may underlie some cardiomyopathies of metabolic origin.

Control of Mitochondrial Substrate Oxidation

Publisher Summary This chapter discusses the control of oxidative metabolism by adenosine diphosphate (ADP) availability in isolated mitochondria and perfused organs. It focuses on the control of dehydrogenase activity. The enzymes, such as, the pyruvate dehydrogenase complex and some of the enzymes of the tricarboxylate cycle have been considered. The most important characteristics of these enzymes are that (1) they catalyze non-equilibrium reactions; (2) exert a dominant influence over the flux through the pathways that are central to catabolic metabolism; and (3) they are mitochondrial. This localization means that the supply of substrate, and dissipation of product, might be limited by the permeability properties of the inner mitochondrial membrane. Because these enzymes are dehydrogenases, the final arbiter of their activity is the respiratory chain, with its coupling to the phosphorylation of ADP. The chapter describes the control of the tricarboxylate cycle and of pyruvate dehydrogenase in their mitochondrial context

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca2+ions. The mechanism is the activation by Ca2+ of four mitochondrial dehydrogenases, viz. glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of ‘downstream’ activation by Ca2+, likely at the level of the FIFo ATPase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca2+ [Ca2+]m which act as a signal to these activities. In this article, we review recent work in which [Ca2+]m is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of [Ca2+]m relative to changes in cytosol free Ca2+ in cells with rapid transients in cytosol Ca2+, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of [Ca2+]m to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, streptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca2+ efflux pathways, thereby raising [Ca2+]m at a given, time-average value of cytosol free Ca2+. (Mol Cell Biochem 184: 359–369, 1997

Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

ABSTRACT The proteins Bcl-2 and Bcl-XL prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-XL in the regulation of cytosolic Ca2+, nitric oxide production (NO), c-Jun NH2-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca2+ATPase, was used to disrupt Ca2+ homeostasis. TG acutely elevated intracellular free Ca2+ and mitochondrial Ca2+ levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca2+ response with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N G-nitro-l-arginine methyl ester hydrochloride (l-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca2+ level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-XL (JT/Bcl-2 or JT/Bcl-XL), NO production, late (36-h) Ca2+ accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-XL cells to the NO donor,S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by l-NAME. Transient expression of a dominant negative mutant SEK1 (Lys→Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-XL inhibited TG-induced loss in mitochondrial membrane potential, release of cytochromec, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca2+ release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca2+-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-XL cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-XL protect the cells against TG-induced apoptosis by negatively regulating Ca2+-sensitive NO synthase activity or expression.

PUFA and aging modulate cardiac mitochondrial membrane lipid composition and Ca2+ activation of PDH

Aberrations in cell Ca2+ homeostasis have been known to parallel both changes in membrane lipid composition and aging. Previous work has shown that the lowered efficiency of work performance, which occurs in isolated hearts from rats fed a diet rich in n-6 polyunsaturated fatty acids (PUFA), relative to those fed n-3 PUFA, could be raised by mitochondrial (Mito) Ca2+ transport inhibition. We tested whether, after Ca2+-dependent stress, the Ca2+-dependent activation of pyruvate dehydrogenase (PDHA/PDHTotal) and Mito Ca2+ cycling could be manipulated by varying the ratio of n-3 to n-6 PUFA in Mito membranes in young (6 mo) and aged (24 mo) isolated rat hearts treated to n-3 or n-6 PUFA-rich diet. Inotropic stimulation by 1 μM norepinephrine (NE) of 24-mo n-6 PUFA-rich hearts elevated total Mito Ca2+ content 38% more than in 6-mo hearts ( P < 0.05). However, both the NE-induced rise in Mito Ca2+ and the difference in response between 6- and 24-mo hearts were partially abolished by n-3 PUFA treatment. NE increased the fractional activation of PDH by 44% above control levels in the 6-mo group compared with 49% in the 24-mo group after n-6 PUFA diet. However, NE stimulation of PDHA was attenuated by n-3 PUFA diet, attaining values only 29 and 23% above control levels in 6- and 24-mo mitochondria, respectively ( P < 0.05). Global ischemia and reperfusion (I/R) in n-6 PUFA hearts gave rise to higher levels of total Mito Ca2+concentration ( P < 0.0001) and PDHA( P < 0.0001) compared with n-3 PUFA. Ruthenium red (3.4 μM) abolished the effects of I/R in all groups. With aging, heart Mito membrane phosphatidylcholine was increased after n-6 PUFA-rich diet (by ∼15%, P < 0.05), whereas cardiolipin and n-3 PUFA content were diminished by 31% ( P < 0.05) and 73% ( P < 0.05), respectively. These effects were prevented by n-3 PUFA-rich diet. The present study, by directly manipulating the cardiac Mito membrane n-3-to-n-6 PUFA ratio, shows that the activation of Ca2+-dependent PDH can be augmented when the n-3-to-n-6 PUFA ratio is low (n-6 PUFA-rich diet; 24-mo hearts) or attenuated when this ratio is relatively high (n-3 PUFA-rich diet). We propose that one of the consequences of dietary-induced manipulation of membrane phospholipids and PUFAs may be the altered flux of Ca2+ across the Mito membrane and thus altered intramitochondrial Ca2+-dependent processes.

An Oxidative Damage-specific Endonuclease from Rat Liver Mitochondria

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE’s molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mm KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mm EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE’s activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.

Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes.

We used left ventricular myocytes from adult rats to investigate the effect of 4 beta-phorbol 12-myristate 13-acetate (PMA) and of sn-1,2-dioctanoylglycerol (DiC-8) on the membrane association of protein kinase C (PKC), cytosolic [Ca2+], (Cai) homeostasis, and the contractile properties of single cardiac cells. Because PKC activity is known to be highly Ca2+ sensitive, the K+ concentration of the bathing medium was raised from 5 to 30 mM in some experiments, a perturbation known to depolarize the cell and increase Cai. In cell suspensions both PMA (3 x 10(-10) and 3 x 10(-7) M) and DiC-8 (10(-5) and 10(-4) M) increased membrane association of PKC. The effect of PMA (10(-7) M) on PKC translocation was enhanced in 30 mM KCl compared with 5 mM KCl. During steady field stimulation at 1 Hz in 1 mM bathing [Ca2+], both PMA (10(-7) M) and DiC-8 (10(-5) M) decreased twitch amplitude to approximately 60% of control in 5 mM KCl, and the negative inotropic effect of either drug was more pronounced in 30 mM KCl than in 5 mM KCl. In single cardiac myocytes loaded with the Ca2+ indicator indo-1 and bathed in 5 mM KCl, we simultaneously measured cell length and Cai. The myofilament responsiveness to Ca2+ was assessed by the relation between contraction amplitude and the peak of the Cai transient. The negative inotropic effect of both PMA and DiC-8 was related to a diminished amplitude of the Cai transient and not to a decreased myofilament responsiveness to Ca2+. In the absence of electrical stimulation, PMA (10(-7) M) and DiC-8 (10(-5) M) decreased the frequency of contractile waves due to spontaneous Ca2+ release from the sarcoplasmic reticulum, and DiC-8 also decreased resting Cai. Thus, activation of PKC, which is thought to occur as part of the response of cardiac muscle to alpha 1-adrenergic stimulation, is associated with a negative inotropic action due to a smaller Cai transient rather than to a decrease in the myofilament responsiveness to Ca2+. These effects on the membrane association of PKC and on contractility are enhanced by cell depolarization achieved by raising [KCl] in the bathing medium.

Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria

Abstract Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 μ m as substrate in the presence or absence of an optimal concentration of palmitoylcarnitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilization has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under Vmax conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial NAD + NADH and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of palmitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcarnitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial NAD + NADH ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca2+ concentration of incubation media from 10−7 to 10−6 m using CaCl2-ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca2+ ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.

Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes

OBJECTIVE Mitochondrial [Ca2+] ([Ca2+]m) rises in parallel with cytosolic [Ca2+] ([Ca2+]c) following ATP-depletion rigor contracture induced by hypoxia in isolated cardiomyocytes. We investigated the pathways involved in the hypoxia induced changes in [Ca2+]m by using known inhibitors of mitochondrial Ca2+ transport, namely ruthenium red, an inhibitor of the Ca2+ uniporter (the normal influx route) and clonazepam, an inhibitor of Na+/Ca2+ exchange, (the normal efflux route). METHODS [Ca2+]m was determined from indo-1/am loaded rat myocytes where the cytosolic fluorescence signal had been quenched by superfusion with Mn2+. [Ca2+]c was measured by loading myocytes with indo-1 pentapotassium salt during the isolation procedure. Cells were placed in a specially developed chamber for induction of hypoxia and reoxygenated 40 min after rigor development. RESULTS 50% of control cells hypercontracted upon reoxygenation; this correlated with a [Ca2+]m or [Ca2+]c higher than approximately 350 nM at the end of rigor. Clonazepam completely abolished the rigor-induced rise in [Ca2+]m but not [Ca2+]c. On reoxygenation [Ca2+]m increased over the first 5 min and remained elevated whereas [Ca2+]c fell. In the presence of ruthenium red a dramatic increase in [Ca2+]m occurred 5-10 min after rigor development (the indo-1 fluorescence signal was saturated); [Ca2+]c also increased but to a lesser extent. On reoxygenation, [Ca2+]m fell rapidly even though cells hypercontracted and [Ca2+]c remained elevated. CONCLUSIONS During hypoxia following rigor development Ca2+ uptake into mitochondria occurs largely via the Na+/Ca2+ exchanger rather than the Ca2+ uniporter whereas on reoxygenation the transporters resume their normal directionality.