Search

We describe a believed-novel procedure for translating metabolite profiles (metabolome) into the set of metabolic fluxes (fluxome) from which they originated. Methodologically, computational modeling is integrated with an analytical platform comprising linear optimization, continuation and dynamic analyses, and metabolic control. The procedure was tested with metabolite profiles obtained from ex vivo mice Langendorff-heart preparations perfused with glucose. The metabolic profiles were analyzed using a detailed kinetic model of the glucose catabolic pathways including glycolysis, pentose phosphate (PP), glycogenolysis, and polyols to translate the glucose metabolome of the heart into the fluxome. After optimization, the ability of the model to simulate the initial metabolite profile was confirmed, and metabolic fluxes as well as the structure of control and regulation of the glucose catabolic network could be calculated. We show that the step catalyzed by phosphofructokinase together with ATP demand and glycogenolysis exert the highest control on the glycolytic flux. The negative flux control exerted by phosphofructokinase on the PP and polyol pathways revealed that the extent of glycolytic flux directly affects flux redirection through these pathways, i.e., the higher the glycolytic flux the lower the PP and polyols. This believed-novel methodological approach represents a step forward that may help in designing therapeutic strategies targeted to diagnose, prevent, and treat metabolic diseases.

Congestive heart failure frequently involves reduced cardiac contractility and slowed muscle relaxation. The reactive nitrogen species HNO (nitroxyl, azanone) has been shown to augment contractility and accelerate relaxation in both normal and failing hearts. In humans, greater than 70% of cytosolic Ca2+ is pumped into the sarco(endo)plasmic reticulum (SR) by SR Ca2+-ATPase (SERCA2a). Phospholamban (PLN) is a small transmembrane protein, which is associated with SERCA2a, and depending on its phosphorylation state, regulates the activity of the Ca2+ pump. Our previous work has shown that HNO modifies PLN and affects SERCA2a activity independent of the β-adrenergic pathway. We have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. These studies demonstrate that PLN cysteines 46 and 41 are the major sites of HNO reactivity. Furthermore, our results indicate that sulfinamide formation is the major HNO-derived modification in the presence of excess HNO donor, whereas disulfide formation is expected to dominate under physiologically relevant conditions.

Selective alpha7 nicotinic receptor agonists, such as GTS-21, are known to have neuroprotective and anti-inflammatory actions, but the effect of these agents on the heart is not known. Therefore, we evaluated the actions of GTS-21 in isolated atria and isolated hearts from C57BL/6 mice. GTS-21 (1-100 μM) caused a concentration-dependent increase in amplitude of contractions and decrease in beating rate in isolated atria. Percent changes in rate and contractile force evoked by 30 μM GTS-21 were −14 ± 2 and +36 ± 11, respectively (n = 4). The onset of responses to GTS-21 was slower than would occur to acetylcholine and norepinephrine, and effects of GTS-21 were not blocked by atropine or atenolol. The impact of GTS-21 on left ventricular function was evaluated next in isolated hearts paced at 400 BPM. Treatment with 30 µM GTS-21 caused significant increases in contractile function: developed pressure (+30 ± 4%); peak systolic pressure (+16 ± 2%); dP/dtmax (+31 ± 6%). Diastolic pressure was also decreased (−60 ± 10%) while the rate of relaxation increased (-dP/dtmax, +36 ± 6%) with no sizable change in coronary perfusion pressure (p < 0.05 for all parameters, n = 4). Surprisingly, responses to GTS-21 were not affected by a selective alpha7 antagonist or the nonselective nicotinic antagonist mecamylamine, and there was no evidence of desensitization. Our data demonstrate that GTS enhances atrial and ventricular contractility and improves LV relaxation with a modest decrease in heart rate. GTS-21 cardiac actions occur by a mechanism that is independent of nicotinic receptors and autonomic nerves. The unique profile of cardiac effects for GTS-21 suggests its potential use in the setting of acute heart failure.

Aims: Abnormal energy metabolism contributes to heart failure (HF) and the failing heart is energy starved. Here we tested whether augmented CK energy metabolism improves myocyte dysfunction in experimental HF.Methods and Results: We tested the response to the β-agonist isoproterenol (2.5 nM, ISO) in cardiomyocytes isolated from wild-type (WT) mice and mice over-expressing cardiac myofibrillar and mitochondrial CK (CK-M and CK-mito) from sham and HF (8 wk transverse aortic constriction, TAC) hearts, to dissect whether over-expressing CK-M or CK-mito might alter myocyte function at baseline or after an increase in energetic demand. At baseline, there were no differences in sarcomere fractional shortening (FS) or whole Ca2+ transient amplitude in response to ISO among sham WT, CK-M or CK-mito myocytes. However, ISO impact on FS, Ca2+ transient, time to 50 Ca2+ decay, and sarcomere re-lengthening were all reduced in WT TAC hearts, consistent with prior reports. Conversely, over-expressing CK-M or CK-mito rescued ISO-induced inotropy in TAC myocytes. No sizable differences in ISO response were noticed in cells obtained from sham WT, CK-M or CK-mito hearts. To test whether over-expressing CK-M or CK-mito confers a degree of protection against acute oxidative stress, non-TAC myocytes were exposed to H2O2 (50 μM for 10 min). The interval between the beginning of H2O2 superfusion and the appearance of an irreversible arrhythmia was measured. WT and CK-M myocytes showed a similar response (359±87s vs. 370±60s, n=5), whereas in CK-mito this interval was prolonged (580±74s).Conclusions: Over-expressing CK-M and CK-mito under failing-TAC conditions improves myocyte contraction and relaxation, likely through preserved Ca2+ handling; however, only the up-regulation of CK-mito can effectively buffer ROS, especially those of mitochondrial origin.

Hyperglycemia and hyperlipidemia are two main traits of type-2 diabetes (T2DM). T2DM patients may develop a cardiomyopathy, and the excess in nutrients greatly contributes to systolic and diastolic dysfunction. The Randle cycle postulates that fatty acid (FA) utilization further impairs glucose utilization, impeding its oxidation. Yet recent evidence suggests that, when acutely infused, FAs such as palmitate (Palm) actually help in maintaining function in T2DM hearts stressed with high glucose and catecholamines. Thus, under conditions of sustained stress, lipids may be necessary to maintain function in stressed T2DM hearts. Using a novel procedure for translating metabolomics into metabolic fluxes, here we tested whether Palm is able to redirect the glucose fluxome in T2DM hearts, contributing to a better utilization/oxidation of glucose. We found that Palm, without inhibiting glycolysis, led to a 50% increase in glucose oxidation via the pentose phosphate [PP] pathway. Palm presence shifted the control of the glycolytic flux from phosphofructokinase to glucose uptake, glucose 6-phosphate dehydrogenase and glycogenolysis. Palm-induced remodeling of the glucose fluxome decreased the intracellular levels of glucose by 17-fold, owing to reduced uptake at maintained utilization. Moreover, it augmented the content of reduced GSH, via higher NADPH generation through the PP pathway. Our study provides a mechanistic explanation to the in vitro observation that FAs such as Palm are necessary for the T2DM hearts to maintain function when in presence of hyperglycemia and/or increased workload, by remodeling glucose utilization leading to a higher supply of reducing equivalents to the heart. Present findings suggest that in T2DM subjects the Randle cycle may apply to some but not all pathophysiological contexts.

Our enjoyable collaboration with Dr. Jeffrey P. Froehlich focused on the role of phospholamban (PLN) in HNO-induced enhancement of SR Ca2+-ATPase (SERCA2a) activity. We hypothesized that given HNO thiophilic nature it modifies cysteine residues in PLN transmembrane domain, altering its interaction with SERCA2a, thus enhancing pump activity. When HNO, donated by Angeli's salt (AS), was administered to control isolated myocytes, it enhanced both sarcomere shortening and Ca2+ transient. However, when AS/HNO was applied to PLN-/- ventriculocytes, HNO inotropy was reduced by ≅ 50% (the remaining likely stemming from enhanced myofilament sensitivity to Ca2+). PLN centrality to HNO cardiotropic action was confirmed incubating SR vesicles from WT and PLN-/- mice with AS/HNO to measure ATP-dependent Ca2+ uptake by stopped-flow mixing. In WT, HNO increased Ca2+ uptake rate, but it failed to do so in PLN-/- vesicles. The role of cysteines in PLN emerged from studies using ER microsomes from Sf21 insect cells expressing SERCA2a±PLN (WT or Cys 36-41-46→Ala mutant) where we assessed SERCA2a dephosphorylation, a measure of E2P hydrolysis, i.e. a rate-limiting step of SERCA2a activity. AS/HNO augmented SERCA2a dephosphorylation in ER microsomes co-expressing SERCA2a and WT PLN, but this stimulation was absent in microsomes expressing SERCA2a and Cys 36-41-46→Ala mutant PLN. Thus, Jeff's elegant, creative and passionate approach helped us to show that PLN is essential for HNO-induced faster Ca2+ uptake by SERCA2a, suggesting that HNO action occurs, at least partly, via modifications of critical cysteines in PLN transmembrane domain. In Jeff's view, “forbidden” disulfide bonds in PLN are involved, and one of his legacies for us is unearthing the cysteine pairs that are involved in HNO-induced formation of an intramolecular bond that could distort the conformation of PLN, thus perturbing its interaction with SERCA2a and relieving the inhibition.

Background: Excitation-contraction coupling and β-adrenergic activation are altered in diabetic hearts, contributing to contractile dysfunction. We hypothesized that mitochondrial dysfunction in diabetic hearts contributes to altered β-adrenergic responses via increased oxidative stress and respiratory uncoupling. Methods: Basal and isoproterenol (ISO)-induced changes in sarcomere shortening and Ca2+ transients were assessed in cardiomyocytes from wild-type (WT) and db/db mice under euglycemia (5.5mM) or hyperglycemia (30mM). Reactive oxygen species (ROS; H2O2 and O2.-), NADH, and ΔΨm were monitored using two photon laser scanning fluorescence microscopy. Results: Basal fractional shortening (FS) and Ca2+ transients were not significantly different between WT and db/db myocytes, regardless of glucose concentration. Following ISO (10nM), FS increased by ≅150% and Ca2+ transients by ≅30%, in both WT and db/db myocytes under euglycemia. Under hyperglycemia, the WT ISO response was intact, but the increase in FS and Ca2+ transients was blunted in db/db cells (68±2%, and 12±3%, respectively, both p

In type 2 diabetes (T2DM), hyperglycemia (HG) and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle's functionality. These perturbations sustain basal low-cardiac performance and limited exercise capacity. We have recently reported that improving the redox/energetic balance of T2DM (db/db) murine cardiomyocytes/hearts with GSH or palmitate (Palm) preserves contractile performance under high-energy demand imposed by combined HG and β-adrenergic stimulation. To further understanding the metabolic basis of Palm salutary action, we first applied metabolomics to Langendorff-perfused T2DM murine hearts subjected to energy/redox stress conditions. In the absence of Palm, HG activates polyol pathways (sorbitol, glycerol, xylitol) triggering flux limitations in glycolytic and pentose phosphate (PP) pathways. The metabolite profile under Palm reveals that this fatty acid (FA) reverses the detrimental action of HG by decreasing polyol accumulation and increasing flux through PP, glycolytic, and beta-oxidation pathways, with a concomitant improvement in cardiomyocyte contraction. To confirm this observation, next we inhibited the polyol or activated the PP pathways. Preincubating HG+ISO-treated myocytes with the aldose reductase inhibitor Zopolrestat (1μM) improved the adrenergic inotropic response, in parallel with the re-activation of glycolysis and oxidative phosphorylation. These same effects were observed pre-incubating HG+ISO challenged myocytes with the activator of transketolase benfotiamine (50μM). Present data indicate that in T2DM hearts HG/ISO regimen unveils a status of mitochondrial dysfunction that favors adverse intracellular redox/energetic conditions and a metabolic remodeling that implies a glucose shunt to polyol pathways and an apparent blockade of glycolysis. These changes appear to be relieved by the exogenous administration of the FA Palm.

Diabetes is a syndrome in which mitochondrial alterations may participate in the dysfunction, and ultimately failure of several organs. The diabetic heart in particular can be subjected to both glycemic level oscillations and increased workload (via β-stimulation), events that may further deteriorate its mechanical properties. Here we show that when diabetic heart (db/db) cells are challenged with high levels of extracellular glucose (30 mM) and concomitant β-adrenergic stimulation via isoproterenol (ISO), intracellular GSH is greatly diminished while superoxide and H2O2 generation substantially increased. The typical enhancement in sarcomere shortening and whole Ca2+ transient driven by β-adrenergic stimulation is blunted in db/db cells, along with hampered relaxation and Ca2 reuptake. Mitochondria isolated from db/db hearts subjected to high-glucose/ISO regimen exhibit markedly decreased coupling ratios from respiratory complexes I, II, and IV while ROS emission is greatly increased, under both forward and reverse electron transport. These changes reflect a profound alteration in the crucial (energy supplying) state 4→3 transition that ultimately results in lowered ATP synthesis and in a “non-stop” reactive oxygen species (ROS) emission in the presence of ADP. Incubating high-glucose/ISO treated db/db myocytes with cell-permeable GSH restores intracellular GSH, blunts ROS production and fully rescues contractility/relaxation in these myocytes. These results show the direct role played by an oxidized redox environment in triggering the negative synergy among mitochondrial ROS and energetics leading to the E-C coupling impairment. Our study maps mitochondrial sites, i.e. state 4→3 transition, that in diabetic heart cells account, at least in part, for excess ROS emission and loss in energy supply when metabolically and energetically challenged.

Phospholamban (PLN) is an integral membrane protein that regulates the Ca2+ pump (SERCA2a) in cardiac sarcoplasmic reticulum (CSR). Phosphorylation of PLN in response to β-adrenergic stimulation enhances cardiac inotropy by increasing CSR Ca2+ uptake. Nitroxyl (HNO), a new candidate drug therapy for congestive heart failure, improves overall cardiovascular function by increasing Ca2+ release and re-uptake in CSR through a direct interaction with RyR2 and SERCA2a, respectively. Using insect cell ER microsomes expressing SERCA2a +/− PLN (WT and Cys → Ala mutant) we have shown that activation of SERCA2a by HNO is PLN-dependent and entails covalent modification of PLN cysteines. Although HNO stimulates SERCA2a activity by uncoupling PLN from SERCA2a, the role of the cysteine residues in the activation mechanism is not completely understood. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46), affecting the regulatory potency of PLN toward SERCA2a. Examples include intra-molecular disulfide cross-links within single PLN molecules or inter-molecular disulfide cross-links between PLN molecules or PLN and SERCA2a. To test this hypothesis, we have constructed a series of PLN mutants containing single, double and triple cysteine substitutions (alanine replacing cysteine). Each of these mutant PLNs will be co-expressed with SERCA2a in insect cells and cell microsomes will be treated with Angeli's salt (an HNO donor) to determine which cysteine residue(s) are essential for activation monitored by enzyme assay and fluorescence spectroscopy of SERCA2a. The results show that intermolecular PLN disulfides play a minor role in activation by HNO. Studies with the Cys → Ala mutations will be useful in determining which cysteine pairs in PLN contribute to intramolecular disulfide cross-links leading to the relief of PLN inhibition and SERCA2a activation.

HNO donors enhance cardiac inotropy by increasing SR Ca2+ re-uptake/release. Given its thiophylic nature, HNO likely modifyies critical cysteine residues in E-C coupling proteins. Phospholamban (PLN) is a potential target for HNO, and its genetic removal or mutation of PLN cysteines should abolish/blunt HNO cardiac effects. Cardiomyocytes were isolated from PLN knockout (PLN-/-) and wildtype (WT) mice, field-stimulated and assessed for Ca2+ transients and sarcomere shortening (SS). HNO effects on the SR-Ca2+ATPase (SERCA2a) were evaluated by isolating SR vesicles from PLN-/- and WT mice and measuring Ca2+ uptake by stopped-flow mixing. Dephosphorylation of SERCA2a (a measure of E2P hydrolysis) was investigated in ER microsomes from Sf21 insect cells expressing SERCA2a±PLN (WT or Cys 36-41-46->Ala mutant). PLN-/- myocytes showed enhanced myocyte contraction and a blunted response to isoproterenol. When challenged with the HNO donor Angeli's salt (AS, 0.5 mM), Ca2+ transient amplitude (-15±5 vs 17±7% in WT, p Ala PLN (0.21 vs 0.18 s-1). We conclude that PLN is essential for the HNO-mediated increase in Ca2+ uptake by SERCA2a, and that modification of PLN thiols is central to this modulation. Enhancing Ca2+ uptake by HNO may benefit heart failure patients that often display depressed SR function.

Respiring mitochondria continually produce hydrogen peroxide (H2O2). When production exceeds scavenging capacity, increased H2O2 emission will occur, thus transforming potential signaling into overt oxidative stress and cellular dysfunction. Glutathione and thioredoxin (Trx) are two major systems providing reducing equivalents, yet their relative contribution in keeping proper mitochondrial redox balance is unclear. Here we hypothesize that under physiological forward electron transport (FET) mode Trx2 is a major ROS buffering system in mitochondria. Thioredoxin reductase 2 (TrxR2) reconverts oxidized Trx2 into its reduced, active form, in mitochondria. Utilizing the highly specific TrxR2 inhibitor Auronofin (AF) we measured H2O2 flux from isolated mitochondria and ventricular myocytes obtained from C57/BL6 mice or guinea pig hearts. In isolated mitochondria, H2O2 emission and the redox status of Trx2 were analyzed in parallel in the absence or presence of AF, under states 4 and 3 of respiration triggered by Glutamate/Malate (G/M, 5mM) and 1mM ADP, respectively. Mitochondria preincubation with AF resulted in 20-fold or 14-fold rise in H2O2 levels with K0.5 of 4.8±0.5 or 5.8±0.6 nM in states 4 or 3 respiration, respectively, In energized and actively phosphorylating mitochondria we measured a 56±8% rise in reduced Trx2 abundance, equivalent to an increase in reductive potential from −330±4 to −348±6 mV, which could be fully prevented by preincubation with 100nM AF. Importantly, AF had no effect on mitochondrial membrane potential, NADH, or GSH levels, reinforcing the high selectivity of AF while demonstrating the major H2O2 scavenging role played by the Trx2 system. In keeping with the results observed in isolated mitochondria, administering AF to cardiomyocytes provoked a rise in total cellular O2.- and H2O2 levels, resulting in reduced cell contractility. Our data indicate that the Trx2 system controls mitochondrial redox balance, H2O2 emission, and ultimately cardiomyocyte function.

The heart from a diabetic animal exhibits dysfunction when exposed to challenging energetic and tissue redox conditions. We showed that when cardiomyocytes from type-2 diabetes (db/db) mice are exposed to high glucose (HG) and β-adrenergic stimulation (via isoproterenol, ISO), these cells show blunted β-contractile reserve and increased oxidative stress. Treating these cells with the fatty acid (FA) palmitate (Palm) offsets those changes, an effect likely due to its ability of generating more reducing equivalents. Yet whether Palm infusion may benefit contractile performance and vascular tone of db/db hearts subjected to metabolic/redox stress is unclear. Using a Langendorff approach, we perfused wild type (WT) and db/db mice with HG (30mM glucose) + ISO (10nM), in absence or presence of Palm. WT hearts were infused with 0.2mM Palm and db/db ones with 0.4mM Palm to mimic higher circulating FA content in diabetic animals. Under HG, coronary perfusion pressure (CPP) was higher in db/db hearts (92±8 vs. 63±7 mmHg, p