Hexokinase II and reperfusion injury: TAT-HK2 peptide impairs vascular function in Langendorff-perfused rat hearts.

RATIONALE: Mitochondrial-bound hexokinase II (HK2) was recently proposed to play a crucial role in the normal functioning of the beating heart and to be necessary to maintain mitochondrial membrane potential. However, our own studies confirmed that mitochondria from ischemic rat hearts were HK2-depleted, yet showed no indication of depolarization and responded normally to ADP. OBJECTIVE: To establish whether the human TAT-HK2 peptide used to dissociate mitochondrial-bound HKII in the Langendorff-perfused heart may exert its effects indirectly by impairing coronary function. METHODS AND RESULTS: Ischemic preconditioning was blocked in rat hearts perfused with 2.5 µmol/L TAT-HK2 before ischemia or at the onset of reperfusion. However, TAT-HK2 also decreased the phosphocreatine:ATP ratio that correlated with reduced rate pressure product and increased diastolic pressure. These effects were preceded by increased aortic pressure (Langendorff constant flow) or decreased coronary flow (Langendorff constant pressure), which was also observed, albeit less pronounced, at 200 nmol/L TAT-HK2 and was prevented by coperfusion with the NO-donor diethylamine NONOate. Mitochondria from TAT-HK2-perfused hearts showed no loss of bound HK2, unlike mitochondria from ischemic hearts where the expected loss was prevented by ischemic preconditioning. CONCLUSIONS: In the perfused rat heart, TAT-HK2 should be used with caution and careful attention to dosage because some of its effects may be mediated by vasoconstriction of the coronary vasculature rather than dissociation of HK2 from myocyte mitochondria.

Regulation of ATP production by mitochondrial Ca(2+).

Stimulation of mitochondrial oxidative metabolism by Ca(2+) is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca(2+) regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic ß-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca(2+)-dependent regulation of ATP levels dynamically in single cells.

Mitochondria and heart disease.

Mitochondria play a key role in the normal functioning of the heart, and in the pathogenesis and development of various types of heart disease. Physiologically, mitochondrial ATP supply needs to be matched to the often sudden changes in ATP demand of the heart, and this is mediated to a large extent by the mitochondrial Ca(2+) transport pathways allowing elevation of mitochondrial [Ca(2+)] ([Ca(2+)](m)). In turn this activates dehydrogenase enzymes to increase NADH and hence ATP supply. Pathologically, [Ca(2+)](m) is also important in generation of reactive oxygen species, and in opening of the mitochondrial permeability transition pore (MPTP); factors involved in both ischaemia-reperfusion injury and in heart failure. The MPTP has proved a promising target for protective strategies, with inhibitors widely used to show cardioprotection in experimental, and very recently human, studies. Similarly mitochondrially-targeted antioxidants have proved protective in various animal models of disease and await clinical trials. The mitochondrial Ca(2+) transport pathways, although in theory promising therapeutic targets, cannot yet be targeted in human studies due to non-specific effects of drugs used experimentally to inhibit them. Finally, specific mitochondrial cardiomyopathies due to mutations in mtDNA have been identified, usually in a gene for a tRNA, which, although rare, are almost always very severe once the mutation has exceeded its threshold.

The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia.

Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H2O2 to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging.

Controlled hyperkalemic reperfusion with magnesium rescues ischemic juvenile hearts by reducing calcium loading.

OBJECTIVES: Our objectives were (1) to determine whether elevated Mg(2+) in controlled hyperkalemic reperfusate without intervention during ischemia protects the juvenile heart against reperfusion injury; and (2) to identify the mechanism(s) underlying any protective effect of Mg(2+). METHODS: Langendorff-perfused hearts from juvenile (11- to 14-day-old) guinea pigs were subjected to mild (30-minute) or severe (45-minute) normothermic global ischemia and 35-minute reperfusion. Hearts were subjected to controlled hyperkalemic reperfusion without or with various concentrations of Mg(2+) (5, 10, 16, 23 mM). The mechanisms underlying the effect of Mg(2+) on intracellular Ca(2+) ([Ca(2+)]i) were also studied in isolated cardiomyocytes exposed to metabolic inhibition followed by washout using hyperkalemic solutions (reperfusion). RESULTS: Sixteen mM Mg(2+) conferred maximal cardioprotection as assessed by improved functional recovery and reduced cardiac injury; this was associated with a significant recovery of cardiac energetics and metabolism following both mild and severe ischemia. The Mg(2+)-induced protection was additive to that of hyperkalemia following mild ischemia and conferred protection following severe ischemia when hyperkalemia alone had no significant effect. Elevated Mg(2+) in the hyperkalemic reperfusate of cardiomyocytes acutely prevented [Ca(2+)]i loading following mild metabolic inhibition and augmented the fall in [Ca(2+)]i following severe metabolic inhibition. CONCLUSIONS: This work demonstrates for the first time in juvenile hearts that elevated Mg(2+) during controlled hyperkalemic reperfusion rescues the heart following ischemia, and that this is likely to be facilitated by reducing [Ca(2+)]i which, in turn, would aid metabolic recovery.

The ups and downs of mitochondrial calcium signalling in the heart.

Regulation of intramitochondrial free calcium ([Ca2+]m) is critical in both physiological and pathological functioning of the heart. The full extent and importance of the role of [Ca2+]m is becoming apparent as evidenced by the increasing interest and work in this area over the last two decades. However, controversies remain, such as the existence of beat-to-beat mitochondrial Ca2+ transients; the role of [Ca2+]m in modulating whole-cell Ca2+ signalling; whether or not an increase in [Ca2+]m is essential to couple ATP supply and demand; and the role of [Ca2+]m in cell death by both necrosis and apoptosis, especially in formation of the mitochondrial permeability transition pore. The role of [Ca2+]m in heart failure is an area that has also recently been highlighted. [Ca2+]m can now be measured reasonably specifically in intact cells and hearts thanks to developments in fluorescent indicators and targeted proteins and more sensitive imaging technology. This has revealed interactions of the mitochondrial Ca2+ transporters with those of the sarcolemma and sarcoplasmic reticulum, and has gone a long way to bringing the mitochondrial Ca2+ transporters to the forefront of cardiac research. Mitochondrial Ca2+ uptake occurs via the ruthenium red sensitive Ca2+ uniporter (mCU), and efflux via an Na+/Ca2+ exchanger (mNCX). The purification and cloning of the transporters, and development of more specific inhibitors, would produce a step-change in our understanding of the role of these apparently critical but still elusive proteins. In this article we will summarise the key physiological roles of [Ca2+]m in ATP production and cell Ca2+ signalling in both adult and neonatal hearts, as well as highlighting some of the controversies in these areas. We will also briefly discuss recent ideas on the interactions of nitric oxide with [Ca2+]m.

Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells.

Mitochondrial Ca(2+) transport was initially considered important only in buffering of cytosolic Ca(2+) by acting as a "sink" under conditions of Ca(2+) overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca(2+) ([Ca(2+)](m)) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a "parallel activation model" whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca(2+)] that was relayed by the mitochondrial Ca(2+) transporters into the matrix to give an increase in [Ca(2+)](m). This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca(2+)](m) in living cells that proved [Ca(2+)](m) changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca(2+)], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca(2+)] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca(2+) is finely tuned to the demands and function of the individual organ.

Mitochondrial calcium transport in the heart: physiological and pathological roles.

That intramitochondrial free calcium ([Ca(2+)](m)) plays various critical roles in both normal physiological and pathological conditions in the heart is now well-accepted, and evidenced by the interest and work in this area of the last two decades. However, controversies remain; such as the existence of beat-to-beat mitochondrial Ca(2+) transients, role of [Ca(2+)](m) in modulating whole-cell Ca(2+) signalling, whether or not [Ca(2+)](m) is critical for increases in ATP supply upon increased demand, and its role in cell death by both necrosis and apoptosis, especially in formation of the mitochondrial permeability transition pore and in ischaemic preconditioning. Neither is there a consensus as to whether inhibiting the Ca(2+) influx or efflux pathways--the Ca(2+) uniporter (MCU) and Na(+)/Ca(2+)-excahnger (mNCX), respectively--is cardioprotective, largely due to lack of specific inhibitors of these transporters. Ruthenium red, Ru360, clonazepam and CGP37157 are all very effective in isolated mitochondria, but reports of their effectiveness in whole cell and heart studies vary considerably, which partly accounts for the lack of a consensus on protective effects. The purification and cloning of the transporters, and development of more specific inhibitors, would produce a step-change in our understanding of the role of these apparently critical but still elusive proteins. However, developments in fluorescent indicators, proteins and imaging technology have meant that [Ca(2+)](m) can now be measured reasonably specifically in intact cells and hearts, and interactions of the mitochondrial Ca(2+) transporters with those of the sarcolemma or sarcoplasmic reticulum are being revealed. This has gone a long way to bringing the transporters to the forefront of cardiac research.

ATP regulation in adult rat cardiomyocytes: time-resolved decoding of rapid mitochondrial calcium spiking imaged with targeted photoproteins.

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca(2+) in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP](m)) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP](c)) were much smaller (<5%), indicating that [ATP](c) was effectively buffered in this compartment. Although mitochondrial [Ca(2+)] ([Ca(2+)](m)) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca(2+) transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP](m) occurred over the same time scale as average [Ca(2+)](m) was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca(2+)](m) were observed at 0.9 or 1.3 mum, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca(2+) transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.

Glutamate loading protects freshly isolated and perfused adult cardiomyocytes against intracellular ROS generation.

Glutamate loading has been shown to protect single isolated perfused cardiomyocytes against metabolic inhibition and wash-off. The mechanism underpinning this protection is unknown. This study aimed to investigate whether reactive oxygen species (ROS) are generated by single isolated perfused cardiomyocytes and whether the protective effect of glutamate loading on cell metabolism is linked to ROS. Single rat cardiomyocytes were isolated with or without glutamate to stimulate glutamate loading. ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate in various stressful conditions including metabolic inhibition and wash-off with/without antimycin A or myxothiazol; simulated ischaemia (without cyanide) and glucose reintroduction; and H(2)O(2) perfusion. Reduced glutathione (GSH) levels were measured in control and glutamate-loaded cells with/without exposure to H(2)O(2). Finally, the effect of glutamate on glutathione reductase and glutathione peroxidase activity was measured. In every stressful condition studied, ROS production was significantly lower in glutamate-loaded cells compared to controls. This occurred regardless of whether ROS were produced intracellularly (e.g. from the respiratory chain inhibited with antimycin A) or via the extracellular precursor H(2)O(2). Glutamate-loaded cells also maintained their morphological integrity at higher H(2)O(2) concentrations than control cells. Furthermore, during H(2)O(2) exposure GSH levels decreased in glutamate-loaded cells but stayed constant in control cells. Glutamate stimulated the activity of glutathione peroxidase in a concentration-dependent fashion. These results provide new evidence to show that the cardioprotective effect of glutamate loading may be mediated through an enhanced ability to destroy ROS in the cell.

Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart.

Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia. Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery. Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening. MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[3H]glucose ([3H]DOG) and measurement of mitochondrial [3H]DOG entrapment. We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover. However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress. Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release). However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening. Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly.