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.

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.

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.

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".

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.

Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator.

The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.

A fast black-blood sequence for four-dimensional cardiac manganese-enhanced MRI in mouse.

The increasing number of mouse models of cardiac diseases requires improvements in the current MRI tools. Anatomic and functional cardiac phenotyping by MRI calls for both time and space resolution in three dimensions. Black-blood contrast is often needed for the accurate delineation of myocardium and chambers, and is consistent with manganese contrast enhancement. In this article, we propose a fast, three-dimensional, time-resolved (four-dimensional), black-blood MRI sequence that allows mouse heart imaging at 10 periods of the cardiac cycle within 30 min at an isotropic resolution of 200 µm. Two-dimensional imaging was possible within 80 s. Blood cancellation was achieved by employing bipolar gradients without the use of a double inversion recovery preparation scheme. Saturation slices were added in two-dimensional experiments for better blood nulling. The rapidity of the two-dimensional acquisition protocol allowed the measurement of the time course of contrast enhancement on manganese infusion. Owing to the very high contrast-to-noise ratio, manganese-enhanced MRI in four dimensions made possible the accurate assessment of regional cardiac volumes in healthy animals. In experimentally infarcted mice, the size of the ischemic zone could be measured easily with this method. The technique might be valuable in evaluating mouse heart diseases and their follow-up in longitudinal studies.

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.

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.

In vivo MR angiography and velocity measurement in mice coronary arteries at 9.4 T: assessment of coronary flow velocity reserve.

PURPOSE: To demonstrate the feasibility of coronary magnetic resonance (MR) angiography in living mice and to evaluate a dynamic MR angiographic method for coronary flow measurement at 9.4-T field strength. MATERIALS AND METHODS: This study was conducted according to European law and was in full compliance with National Institutes of Health recommendations for animal care and a local institutional animal care committee. Mice were anesthetized by using isoflurane. First, time-of-flight MR angiography was performed in 10 mice to measure coronary diameters at 80-mum isotropic resolution. Second, left coronary artery (LCA) velocity measurements were performed at seven cardiac phases in nine other mice to assess the velocity curve profile. Third, coronary velocities were measured at the middiastolic phase in 13 mice at rest and during adenosine-induced hyperemia to calculate coronary flow velocity reserve (CFVR). The Pearson coefficient compared the correlation between isoflurane dose and CFVR. Paired t tests compared R-R intervals and respiratory rates between rest and hyperemia. RESULTS: Proximal diameters were, respectively, 404 mum +/- 34 [standard deviation] and 259 mum +/- 22 for the LCAs and the right coronary arteries, which were in accordance with reported values. The velocity curve profile throughout the cardiac cycle was similar to values from the literature. Baseline and hyperemic velocities were, respectively, 19.0 cm/sec +/- 4.4 and 33.7 cm/sec +/- 4.7 (P<.001), resulting in a CFVR of 1.77 +/- 0.19. CFVR did not correlate with isoflurane dose (r = 0.05, P = .88). R-R intervals shortened by 2.5% during hyperemia (P = .04). Respiratory rates showed no difference between rest and hyperemia (P = .39). CONCLUSION: High-spatial-resolution three-dimensional coronary MR angiography is feasible in living mice. Dynamic MR angiography depicts coronary velocity changes throughout the cardiac cycle and between rest and maximum hyperemia, providing a tool for CFVR assessment.

System analysis of the effect of various drugs on cardiac contraction energetics.

We used MoCA (Modular Control and Regulation Analysis) to demonstrate in intact beating rat heart that physiological activation of contraction by adrenaline involves the almost perfect parallel activation of both mitochondria and myofibrils by intracellular Ca(2+). This explains the perfect homoeostasis of the energetic intermediate PCr (phosphocreatine) in heart. When using drugs specifically stimulating either supply or demand activities, MoCA helped reveal the very specific mode of regulation of heart contraction energetics. Only activation of myofibrils activity (demand), either by increasing intracellular Ca(2+) concentration or myofibrils sensitivity to Ca(2+), triggers activation of contractile activity. In contrast, the activation of mitochondrial activity (supply) has strictly no effect on contraction, either directly or through PCr changes (intermediate).

Absence of mitochondrial activation during levosimendan inotropic action in perfused paced guinea pig hearts as demonstrated by modular control analysis.

Levosimendan is a calcium sensitizer developed for the treatment of heart failure. It increases contractile force by enhancing the sensitivity of myofilaments to calcium. Besides this sensitizing effect, the drug has also been reported to show some inhibitory action on phosphodiesterase 3 (PDE3). The inotropic effects of levosimendan have been studied on guinea pig paced perfused hearts by using modular control analysis (MoCA) (Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiaudiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007.), an integrative approach of heart energetics using noninvasive (31)P NMR. The aim was to evaluate quantitatively the respective effects of this drug on energy supply and demand modules. Under our experimental conditions, 0.7 muM levosimendan induced a 45% increase in paced heart output associated with a 7% decrease in phosphocreatine and a negligible increase in oxygen consumption. Because MoCA allows in situ study of the internal regulations in intact beating heart energetics, it was applied to describe quantitatively by which routes levosimendan exerts its inotropic action. MoCA demonstrated the absence of any significant effect of the drug on the supply module, which is responsible for the lower increase in oxygen consumption, compared with epinephrine, which increases the ratio between myocardial oxygen consumption and cardiac contraction. This result evidences that, under our conditions, a possible effect of levosimendan on PDE3 activity and/or intracellular calcium remains very low on mitochondrial activity and insignificant on integrated cardiac energetics. Thus, levosimendan inotropic effect on guinea pig heart depends almost entirely on the calcium-sensitizing properties leading to myofilament activation and the concomitant activation of energy supply by the decrease in PCr, therefore improving energetic efficiency of contraction.

Effect of pyruvate, lactate and insulin on ATP supply and demand in unpaced perfused rat heart.

Mitochondrial respiration/oxidative phosphorylation is the main source of energy, in the form of ATP, in the heart under physiological conditions. Different respiratory substrates were used in various experiments during heart perfusion: glucose, pyruvate, lactate, glucose+pyruvate, glucose+lactate, glucose+insulin etc. Also under physiological conditions, the concentration of respiratory substrates/hormones in blood can vary significantly. In the present study, we tested the effect of pyruvate, lactate and insulin (all in the presence of glucose) and glucose (in the presence of pyruvate) on the ATP-producing and -consuming blocks in perfused rat heart, in a system where HR (heart rate) was allowed to vary (no pacing). Changes in RPP (rate-pressure product) and PCr (phosphocreatine) concentration were measured. PAA (Proportional Activation Approach) was used to visualize and quantitatively analyse the data. It was demonstrated that addition of glucose (in the presence of pyruvate) exerted essentially no effect on the system. Insulin (in the presence of glucose) activated only the ATP producer. The most interesting finding is that, in our system, pyruvate and lactate (added in the presence or instead of glucose) activated ATP producer, but significantly inhibited ATP consumer (their effect was quantitatively identical).

4D retrospective black blood trueFISP imaging of mouse heart.

The purpose of this study was to demonstrate the feasibility of steady-state True fast imaging with steady precession (TrueFISP) four-dimensional imaging of mouse heart at high resolution and its efficiency for cardiac volumetry. Three-dimensional cine-imaging of control and hypoxic mice was carried out at 4.7 T without magnetization preparation or ECG-triggering. The k-space lines were acquired with the TrueFISP sequence (pulse repetition time/echo time = 4/2 ms) in a repeated sequential manner. Retrospective reordering of raw data allowed the reconstruction of 10 three-dimensional images per cardiac cycle. The acquisition scheme used an alternating radiofrequency phase and sum-of-square reconstruction method. Black-blood three-dimensional images at around 200 mum resolution were produced without banding artifact throughout the cardiac cycle. High contrast to noise made it possible to estimate cavity volumes during diastole and systole. Right and left ventricular stroke volume was significantly higher in hypoxic mice vs controls (20.2 +/- 2 vs 15.1 +/- 2; P < 0.05, 24.9 +/- 2 vs 20.4 +/- 2; P < 0.05, respectively). In conclusion, four-dimensional black-blood TrueFISP imaging in living mice is a method of choice to investigate cardiac abnormalities in mouse models.

Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply.

During low-to-high work transition in adult mammalian heart in vivo the concentrations of free ADP, ATP, PCr (phosphocreatine), P(i) and NADH are essentially constant, in striking contrast with skeletal muscle. The direct activation by calcium ions of ATP usage and feedback activation of ATP production by ADP (and P(i)) alone cannot explain this perfect homoeostasis. A comparison of the response to adrenaline (increase in rate-pressure product and [PCr]) of the intact beating perfused rat heart with the elasticities of the PCr producer and consumer to PCr concentration demonstrated that both the ATP/PCr-producing block and ATP/PCr-consuming block are directly activated to a similar extent during physiological heart activation. Our finding constitutes a direct evidence for the parallel-activation mechanism of the regulation of oxidative phosphorylation in heart postulated previously in a theoretical way.