Real-time resolution studies of the regulation of lactate production by hexokinases binding to mitochondria in single cells.

During hypoxia accumulation of lactate may be a key factor in acidosis-induced tissue damage. Binding of hexokinase (HK) to the outer membrane of mitochondria may have a protective effect under these conditions. We have investigated the regulation of lactate metabolism by hexokinases (HKs), using HEK293 cells in which the endogenous hexokinases have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were also transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown HEK cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. Upon inhibition of the mitochondrial electron transfer chain by NaCN this effect was reversed as a rapid increase in lactate developed which was followed by a slow and sustained increase in the continued presence of the inhibitor. Incubation of the HKI/HKII double knockdown HEK cells with the inhibitor of the malic enzyme, ME1*, blocked the delayed accumulation of lactate evoked by NaCN. With replacement by overexpression of HKI or HKII the accumulation of intracellular lactate evoked by NaCN was prevented. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN) abolished the protective effect of HK expression, with NaCN causing again a sustained increase in lactate. The effect of HK was dependent on HK's catalytic activity and interaction with the mitochondrial outer membrane (MOM). Based on these data we propose that transformation of glucose into G6P by HK activates the pentose phosphate pathway which increases the production of NADPH, which then blocks the activity of the malic enzyme to transform malate into pyruvate and lactate.

Real-time resolution studies of the regulation of pyruvate-dependent lactate metabolism by hexokinases in single cells.

Lactate is a mitochondrial substrate for many tissues including neuron, muscle, skeletal and cardiac, as well as many cancer cells, however little is known about the processes that regulate its utilization in mitochondria. Based on the close association of Hexokinases (HK) with mitochondria, and the known cardio-protective role of HK in cardiac muscle, we have investigated the regulation of lactate and pyruvate metabolism by hexokinases (HKs), utilizing wild-type HEK293 cells and HEK293 cells in which the endogenous HKI and/or HKII have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. This decrease was rapidly reversed upon inhibition of the malate aspartate shuttle by aminooxyacetate, or inhibition of mitochondrial oxidative respiration by NaCN. These results suggest that in the absence of HKs, pyruvate-dependent activation of the TCA cycle together with the malate aspartate shuttle facilitates lactate transformation into pyruvate and its utilization by mitochondria. With replacement by overexpression of HKI or HKII the cellular response to pyruvate and NaCN was modified. With either hexokinase present, both the decrease in lactate due to the addition of pyruvate and the increase following addition of NaCN were either transient or suppressed altogether. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN), abolished the effects of HK replacement. These results suggest that blocking of the malate aspartate shuttle by HK may involve activation of the pentose phosphate pathway and increased NADPH production.

Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.

Integrative Methods for Studying Cardiac Energetics.

The more recent studies of human pathologies have essentially revealed the complexity of the interactions involved at the different levels of integration in organ physiology. Integrated organ thus reveals functional properties not predictable by underlying molecular events. It is therefore obvious that current fine molecular analyses of pathologies should be fruitfully combined with integrative approaches of whole organ function. It follows that an important issue in the comprehension of the link between molecular events in pathologies and whole organ function/dysfunction is the development of new experimental strategies aimed at the study of the integrated organ physiology. Cardiovascular diseases are a good example as heart submitted to ischemic conditions has to cope both with a decreased supply of nutrients and oxygen, and the necessary increased activity required to sustain whole body-including the heart itself-oxygenation.By combining the principles of control analysis with noninvasive 31P NMR measurement of the energetic intermediates and simultaneous measurement of heart contractile activity, we developed MoCA (for Modular Control and regulation Analysis), an integrative approach designed to study in situ control and regulation of cardiac energetics during contraction in intact beating perfused isolated heart (Diolez et al., Am J Physiol Regul Integr Comp Physiol 293(1):R13-R19, 2007). Because it gives real access to integrated organ function, MoCA brings out a new type of information-the "elasticities," referring to integrated internal responses to metabolic changes-that may be a key to the understanding of the processes involved in pathologies. MoCA can potentially be used not only to detect the origin of the defects associated with the pathology, but also to provide the quantitative description of the routes by which these defects-or also drugs-modulate global heart function, therefore opening therapeutic perspectives. This review presents selected examples of the applications to isolated intact beating heart that evidence different modes of energetic regulation of cardiac contraction. We also discuss the clinical application by using noninvasive 31P cardiac energetics examination under clinical conditions for detection of heart pathologies.

The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

The emergence of egalitarianism in a model of early human societies.

How did egalitarianism emerge in early human societies? In contrast to dominance hierarchies in non-human primates, human simple forager bands are typically egalitarian, with male hunters often serving as the collective alpha. Here we present a thermodynamics-inspired simple population model, based on stochastic optimization of dominance relationships, in which a dominance hierarchy of individuals with exclusively self-centered characteristics (the desire to dominate, resentment at being dominated) transitions spontaneously to egalitarianism as their capacity for language develops. Language, specifically gossip, allows resentment against being dominated to promote the formation of antidominance coalitions which destabilize the alpha position for individuals, leading to a phase transition in which a coalition of the full population suddenly becomes dominant. Thus, egalitarianism emerges suddenly as the optimal power-sharing arrangement in a population of selfish individuals without any inherently altruistic qualities. We speculate that egalitarianism driven by punishment for exhibiting alpha-like behavior may then set the stage for genuinely altruistic traits to propagate as predicted by game theory models. Based on model simulations, we also predict that egalitarianism is a pre-condition for adaptation of tools as weapons. Potential implications for origins of human moral belief systems are discussed.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Real-time imaging of sodium glucose transporter (SGLT1) trafficking and activity in single cells.

The processes controlling targeting of glucose transporters to apical and basolateral membranes of polarized cells are complex and not-well understood. We have engineered SGLT1 and GLUT4 constructs linked to fluorescent proteins to highlight the differences in transporter expression and trafficking, in real time, in different cell types. Activity was assessed in parallel using a FRET glucose sensor. In COS cells and HEK cells, SGLT1 was distributed between the plasma membrane and intracellular compartments, but there was little expression in CHO cells. Trafficking was investigated using the lysosome inhibitors NH4Cl (10 mmol/L) and chloroquine (150 µmol/L) and the proteasome inhibitors MG-262 (1 µmol/L) and lactacystin (5 µmol/L). Lysosome inhibitors caused SGLT1 accumulation into intracellular bodies, whereas proteasome inhibitors induced SGLT1 accumulation in the plasma membrane, even in CHO cells. Our data suggest that a fraction of SGLT1 is rapidly degraded by lysosomes and never reached the plasma membrane; another fraction reaches the membrane and is subsequently degraded by lysosomes following internalization. The latter process is regulated by the ubiquitin/proteasome pathway, acting at a late stage of the lysosomal pathway. Using the cholesterol inhibitor MßCD (3 mmol/L), a dominant negative dynamin (K44A) and caveolin, we showed that SGLT1 internalization is lipid raft-mediated, but caveolin-independent. In contrast, GLUT4 internalization is dynamin-dependent, but cholesterol-independent. The physiological relevance of these data is discussed in terms of differential membrane compartmentalization of the transporters and expression under stress conditions.

Reactive oxygen species production in cardiac mitochondria after complex I inhibition: Modulation by substrate-dependent regulation of the NADH/NAD(+) ratio.

Reactive oxygen species (ROS) production by isolated complex I is steeply dependent on the NADH/NAD(+) ratio. We used alamethicin-permeabilized mitochondria to study the substrate-dependence of matrix NADH and ROS production when complex I is inhibited by piericidin or rotenone. When complex I was inhibited in the presence of malate/glutamate, membrane permeabilization accelerated O2 consumption and ROS production due to a rapid increase in NADH generation that was not limited by matrix NAD(H) efflux. In the presence of inhibitor, both malate and glutamate were required to generate a high enough NADH/NAD(+) ratio to support ROS production through the coordinated activity of malate dehydrogenase (MDH) and aspartate aminotransferase (AST). With malate and glutamate present, the rate of ROS production was closely related to local NADH generation, whereas in the absence of substrates, ROS production was accelerated by increase in added [NADH]. With malate alone, oxaloacetate accumulation limited NADH production by MDH unless glutamate was also added to promote oxaloacetate removal via AST. α-ketoglutarate (KG) as well as AST inhibition also reversed NADH generation and inhibited ROS production. If malate and glutamate were provided before rather than after piericidin or rotenone, ROS generation was markedly reduced due to time-dependent efflux of CoA. CoA depletion decreased KG oxidation by α-ketoglutarate dehydrogenase (KGDH), such that the resulting increase in [KG] inhibited oxaloacetate removal by AST and NADH generation by MDH. These findings were largely obscured in intact mitochondria due to robust H2O2 scavenging and limited ability to control substrate concentrations in the matrix. We conclude that in mitochondria with inhibited complex I, malate/glutamate-stimulated ROS generation depends strongly on oxaloacetate removal and on the ability of KGDH to oxidize KG generated by AST.

A large Rab GTPase encoded by CRACR2A is a component of subsynaptic vesicles that transmit T cell activation signals.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

Increased reactive oxygen species production during reductive stress: The roles of mitochondrial glutathione and thioredoxin reductases.

Both extremes of redox balance are known to cause cardiac injury, with mounting evidence revealing that the injury induced by both oxidative and reductive stress is oxidative in nature. During reductive stress, when electron acceptors are expected to be mostly reduced, some redox proteins can donate electrons to O2 instead, which increases reactive oxygen species (ROS) production. However, the high level of reducing equivalents also concomitantly enhances ROS scavenging systems involving redox couples such as NADPH/NADP+ and GSH/GSSG. Here our objective was to explore how reductive stress paradoxically increases net mitochondrial ROS production despite the concomitant enhancement of ROS scavenging systems. Using recombinant enzymes and isolated permeabilized cardiac mitochondria, we show that two normally antioxidant matrix NADPH reductases, glutathione reductase and thioredoxin reductase, generate H2O2 by leaking electrons from their reduced flavoprotein to O2 when electron flow is impaired by inhibitors or because of limited availability of their natural electron acceptors, GSSG and oxidized thioredoxin. The spillover of H2O2 under these conditions depends on H2O2 reduction by peroxiredoxin activity, which may regulate redox signaling in response to endogenous or exogenous factors. These findings may explain how ROS production during reductive stress overwhelms ROS scavenging capability, generating the net mitochondrial ROS spillover causing oxidative injury. These enzymes could potentially be targeted to increase cancer cell death or modulate H2O2-induced redox signaling to protect the heart against ischemia/reperfusion damage.

Integrative methods for studying cardiac energetics.

The more recent studies of human pathologies have essentially revealed the complexity of the interactions involved at the different levels of integration in organ physiology. Integrated organ thus reveals functional properties not predictable by underlying molecular events. It is therefore obvious that current fine molecular analyses of pathologies should be fruitfully combined with integrative approaches of whole organ function. It follows an important issue in the comprehension of the link between molecular events in pathologies, and whole organ function/dysfunction is the development of new experimental strategies aimed at the study of the integrated organ physiology. Cardiovascular diseases are a good example as heart submitted to ischemic conditions has to cope both with a decreased supply of nutrients and oxygen, and the necessary increased activity required to sustain whole body-including the heart itself-oxygenation.By combining the principles of control analysis with noninvasive (31)P NMR measurement of the energetic intermediates and simultaneous measurement of heart contractile activity, we developed MoCA (for Modular Control and Regulation Analysis), an integrative approach designed to study in situ control and regulation of cardiac energetics during contraction in intact beating perfused isolated heart (Diolez et al., Am J Physiol Regul Integr Comp Physiol 293(1):R13-R19, 2007). Because it gives real access to integrated organ function, MoCA brings out a new type of information-the "elasticities," referring to internal responses to metabolic changes-that may be a key to the understanding of the processes involved in pathologies. MoCA can potentially be used not only to detect the origin of the defects associated with the pathology, but also to provide the quantitative description of the routes by which these defects-or also drugs-modulate global heart function, therefore opening therapeutic perspectives. This review presents selected examples of the applications to isolated intact beating heart and a wider application to cardiac energetics under clinical conditions with the direct study of heart pathologies.

Hexokinases and cardioprotection.

As mediators of the first enzymatic step in glucose metabolism, hexokinases (HKs) orchestrate a variety of catabolic and anabolic uses of glucose, regulate antioxidant power by generating NADPH for glutathione reduction, and modulate cell death processes by directly interacting with the voltage-dependent anion channel (VDAC), a regulatory component of the mitochondrial permeability transition pore (mPTP). Here we summarize the current state-of-knowledge about HKs and their role in protecting the heart from ischemia/reperfusion (I/R) injury, reviewing: 1) the properties of different HK isoforms and how their function is regulated by their subcellular localization; 2) how HKs modulate glucose metabolism and energy production during I/R; 3) the molecular mechanisms by which HKs influence mPTP opening and cellular injury during I/R; and 4) how different metabolic and HK profiles correlate with susceptibility to I/R injury and cardioprotective efficacy in cancer cells, neonatal hearts, and normal, hypertrophied and failing adult hearts, and how these difference may guide novel therapeutic strategies to limit I/R injury in the heart. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Hypothesis on Skeletal Muscle Aging: Mitochondrial Adenine Nucleotide Translocator Decreases Reactive Oxygen Species Production While Preserving Coupling Efficiency.

Mitochondrial membrane potential is the major regulator of mitochondrial functions, including coupling efficiency and production of reactive oxygen species (ROS). Both functions are crucial for cell bioenergetics. We previously presented evidences for a specific modulation of adenine nucleotide translocase (ANT) appearing during aging that results in a decrease in membrane potential - and therefore ROS production-but surprisingly increases coupling efficiency under conditions of low ATP turnover. Careful study of the bioenergetic parameters (oxidation and phosphorylation rates, membrane potential) of isolated mitochondria from skeletal muscles (gastrocnemius) of aged and young rats revealed a remodeling at the level of the phosphorylation system, in the absence of alteration of the inner mitochondrial membrane (uncoupling) or respiratory chain complexes regulation. We further observed a decrease in mitochondrial affinity for ADP in aged isolated mitochondria, and higher sensitivity of ANT to its specific inhibitor atractyloside. This age-induced modification of ANT results in an increase in the ADP concentration required to sustain the same ATP turnover as compared to young muscle, and therefore in a lower membrane potential under phosphorylating-in vivo-conditions. Thus, for equivalent ATP turnover (cellular ATP demand), coupling efficiency is even higher in aged muscle mitochondria, due to the down-regulation of inner membrane proton leak caused by the decrease in membrane potential. In the framework of the radical theory of aging, these modifications in ANT function may be the result of oxidative damage caused by intra mitochondrial ROS and may appear like a virtuous circle where ROS induce a mechanism that reduces their production, without causing uncoupling, and even leading in improved efficiency. Because of the importance of ROS as therapeutic targets, this new mechanism deserves further studies.

Mitochondrial energetics is impaired in vivo in aged skeletal muscle.

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging-related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with (31) P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness ('elasticity') of mitochondrial oxidative phosphorylation to contraction-induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging-related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon.

A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.

The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement) during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.

Non-invasive integrative analysis of contraction energetics in intact beating heart.

The comprehensive study of human pathologies has revealed the complexity of the interactions involved in cardiovascular physiology. The recent validation of system's biology approaches - like our Modular Control and Regulation Analysis (MoCA) - motivates the current interest for new integrative and non-invasive analyses that could be used for medical study of human heart contraction energetics. By considering heart energetics as a supply-demand system, MoCA gives access to integrated organ function and brings out a new type of information, the "elasticities", which describe in situ the regulation of both energy demand and supply by cellular energetic status. These regulations determine the internal control of contraction energetics and may therefore be a key to the understanding of the links between molecular events in pathologies and whole organ function/dysfunction. A wider application to the effects of cardiac drugs in conjunction with the direct study of heart pathologies may be considered in the near future. MoCA can potentially be used not only to detect the origin of the defects associated with the pathology (elasticity analyses), but also to provide a quantitative description of how these defects influence global heart function (regulation analysis) and therefore open new therapeutic perspectives. Several key examples of current applications to intact isolated beating heart are presented in this paper. The future application to human pathologies will require the use of non-invasive NMR techniques for the simultaneous measurement of energy status ((31)P NMR) and heart contractile activity (3D MRI). This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Pro- and antiarrhythmic effects of ATP-sensitive potassium current activation on reentry during early afterdepolarization-mediated arrhythmias.

BACKGROUND: Under conditions promoting early afterdepolarizations (EADs), ventricular tissue can become bi-excitable, that is, capable of wave propagation mediated by either the Na current (INa) or the L-type calcium current (ICa,L), raising the possibility that ICa,L-mediated reentry may contribute to polymorphic ventricular tachycardia (PVT) and torsades de pointes. ATP-sensitive K current (IKATP) activation suppresses EADs, but the effects on ICa,L-mediated reentry are unknown. OBJECTIVE: To investigate the effects of IKATP activation on ICa,L-mediated reentry. METHODS: We performed optical voltage mapping in cultured neonatal rat ventricular myocyte monolayers exposed to BayK8644 and isoproterenol. The effects of pharmacologically activating IKATP with pinacidil were analyzed. RESULTS: In 13 monolayers with anatomic ICa,L-mediated reentry around a central obstacle, pinacidil (50 µM) converted ICa,L-mediated reentry to INa-mediated reentry. In 33 monolayers with functional ICa,L-mediated reentry (spiral waves), pinacidil terminated reentry in 17, converted reentry into more complex INa-mediated reentry resembling fibrillation in 12, and had no effect in 4. In simulated 2-dimensional bi-excitable tissue in which ICa,L- and INa-mediated wave fronts coexisted, slow IKATP activation (over minutes) reliably terminated rotors but rapid IKATP activation (over seconds) often converted ICa,L-mediated reentry to INa-mediated reentry resembling fibrillation. CONCLUSIONS: IKATP activation can have proarrhythmic effects on EAD-mediated arrhythmias if ICa,L-mediated reentry is present.