Miguel Aon - Johns Hopkins University (original) (raw)

Papers by Miguel Aon

Metabolic remodelling of glucose, fatty acid and redox pathways in the heart of type 2 diabetic mice

The Journal of Physiology, Apr 1, 2020

Hearts from type 2 diabetic animals display perturbations in excitation–contraction coupling, imp... more Hearts from type 2 diabetic animals display perturbations in excitation–contraction coupling, impairing myocyte contractility and delaying relaxation, along with altered substrate consumption patterns. Under high glucose and β‐adrenergic stimulation conditions, palmitate can, at least in part, offset left ventricle (LV) dysfunction in hearts from diabetic mice, improving contractility and relaxation while restoring coronary perfusion pressure. Fluxome calculations of central catabolism in diabetic hearts show that, in the presence of palmitate, there is a metabolic remodelling involving tricarboxylic acid cycle, polyol and pentose phosphate pathways, leading to improved redox balance in cytoplasmic and mitochondrial compartments. Under high glucose and increased energy demand, the metabolic/fluxomic redirection leading to restored redox balance imparted by palmitate helps explain maintained LV function and may contribute to designing novel therapeutic approaches to prevent cardiac dysfunction in diabetic patients.

To understand the control and regulation of mitochondrial energy metabolism a generalized matrix ... more To understand the control and regulation of mitochondrial energy metabolism a generalized matrix method of Metabolic Control Analysis has been applied to a computational model of mitochondrial energetics. The computational model of Cortassa et al. (2003) encompasses oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and ion dynamics across the inner mitochondrial membrane. Control of respiration and ATP synthesis fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP synthesis and ATP/ADP transport, as well as by Ca 2+ dynamics. The analysis of flux control coefficients and response coefficients has led to the notion of control by diffuse loops, that points to the regulatory interactions exerted by processes that are mechanistically only indirectly related with each other. The approach we have utilized demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

A Remarkable Adaptive Paradigm Of Heart Performance And Protection Emerges In Response To The Constitutive Challenge Of Marked Cardiac-Specific Overexpression Of Adenylyl Cyclase Type 8

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt ... more Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload (∼30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90α, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide arr...

Function, 2021

ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be ... more ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+: H+ stoichiometry. The excellent agreement between the functional data obtai...

Journal of Cell Science, 1991

The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investig... more The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investigated in starved yeast cells that were metabolizing glucose semianaerobically. Glycolytic oscillations were triggered after a pulse of glucose by inhibition of mitochondrial respiration with KCN, myxothiazol and antimycin A or in mutants in the bel complex (ubiquinol:cytochrome c reductase) that were largely deficient in respiratory capacity. Inhibition of the adenine nucleotide translocator by preincubation with bongkrekic acid also triggered a train of damped sinusoidal oscillations after glucose addition. The oscillations consisted of cycles of reduction and oxidation of the intracellular pool of nicotinamide nucleotides with periods of 45 s to 1 min and amplitudes of 0.8 mM or lower. Preincubation with the uncoupler carbonyl cyamide p-(trifluoromethoxy)phenylhydrazone (FCCP) annihilated cyanide-induced oscillations of NAD(P)H. Evidence for de-energization of mitochondrial membranes in...

Frontiers in Physiology, 2020

Mitochondrial criticality describes a state in which the mitochondrial cardiac network under inte... more Mitochondrial criticality describes a state in which the mitochondrial cardiac network under intense oxidative stress becomes very sensitive to small perturbations, leading from local to cell-wide depolarization and synchronized oscillations that may escalate to the myocardial syncytium generating arrhythmias. Herein, we describe the occurrence of mitochondrial criticality in the chronic setting of a metabolic disorder, type 1 diabetes (T1DM), using a streptozotocin (STZ)-treated guinea pig (GP) animal model. Using wavelet analysis of mitochondrial networks from two-photon microscopy imaging of cardiac myocytes loaded with a fluorescent probe of the mitochondrial membrane potential, we show that cardiomyocytes from T1DM GPs are closer to criticality, making them more vulnerable to cell-wide mitochondrial oscillations as can be judged by the latency period to trigger oscillations after a laser flash perturbation, and their propensity to oscillate. Insulin treatment of T1DM GPs rescued cardiac myocytes to sham control levels of susceptibility, a protective condition that could also be attained with interventions leading to improvement of the cellular redox environment such as preincubation of diabetic cardiac myocytes with the lipid palmitate or a cell-permeable form of glutathione, in the presence of glucose.

Cell metabolism, Jan 6, 2018

The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD, i... more The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD, is elusive. Here, we report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Untargeted metabolite profiling of the liver and metabolic flux analysis of liver-derived cells revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Targeted NAD metabolome analysis in liver revealed depressed expression of NAM salvage in NAM-treated mice, an effect counteracted by higher expression of de novo NAD biosynthetic enzymes. Although neither hepatic NADnor NADPwas boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in ...

PLOS Computational Biology, 2017

Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknow... more Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze β-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO 2) and hydrogen peroxide (H 2 O 2) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H 2 O 2 emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of β-oxidation of long-chain fatty acids and main energyredox processes is able to simulate the relationship between VO 2 and H 2 O 2 emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H 2 O 2 emission as a function of VO 2. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply.

Processes, 2015

The advent of high throughput-omics has made the accumulation of comprehensive data sets possible... more The advent of high throughput-omics has made the accumulation of comprehensive data sets possible, consisting of changes in genes, transcripts, proteins and metabolites. Systems biology-inspired computational methods for translating metabolomics data into fluxomics provide a direct functional, dynamic readout of metabolic networks. When combined with appropriate experimental design, these methods deliver insightful knowledge about cellular function under diverse conditions. The use of computational models accounting for detailed kinetics and regulatory mechanisms allow us to unravel the control and regulatory properties of the fluxome under steady and time-dependent behaviors. This approach extends the analysis of complex systems from description to prediction, including control of complex dynamic behavior ranging from biological rhythms to catastrophic lethal arrhythmias. The powerful quantitative metabolomics-fluxomics approach will help our ability to engineer unicellular and multicellular organisms evolve from trial-and-error to a more predictable process, and from cells to organ and organisms.

2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2011

To understand the control and regulation of mitochondrial energy metabolism a generalized matrix ... more To understand the control and regulation of mitochondrial energy metabolism a generalized matrix method of Metabolic Control Analysis has been applied to a computational model of mitochondrial energetics. The computational model of Cortassa et al. (2003) encompasses oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and ion dynamics across the inner mitochondrial membrane. Control of respiration and ATP synthesis fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP synthesis and ATP/ADP transport, as well as by Ca 2+ dynamics. The analysis of flux control coefficients and response coefficients has led to the notion of control by diffuse loops, that points to the regulatory interactions exerted by processes that are mechanistically only indirectly related with each other. The approach we have utilized demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

Journal of General Microbiology, 1992

Cell synchrony was investigated during glycolytic oscillations in starved yeast cell suspensions ... more Cell synchrony was investigated during glycolytic oscillations in starved yeast cell suspensions at cell densities ranging from 2 x 106-5 x lo7 cells ml-l. Oscillations in NAD(P)H were triggered by inhibition of mitochondria1 respiration when intracellular NAD(P)H had reached a steady state after glucose addition. Before macroscopic damping of the oscillations, individual yeast cells oscillated in phase with the cell population. After oscillations had damped out macroscopically, a significant fraction of the cells still exhibited oscillatory dynamics, slightly out-ofphase. At cell concentrations higher than lo7 cells ml-l the dependence upon cell-density of (i) the damping of glycolytic oscillations and (ii) the amplitude per cell suggested that cell-to-cell interaction occurred. Most importantly, at cell densities exceeding lo7 cells ml-l the damping was much weaker. A combination of modelling studies and experimental analysis of the kinetics of damping of oscillations and their amplitude, with and without added ethanol, pyruvate or acetaldehyde, suggested that the autonomous glycolytic oscillations of the yeast cells depend upon the balance between oxidative and reductive (ethanol catabolism) fluxes of NADH, which is affected by the extracellular concentration of ethanol. Based on the facts that cell (i) excrete ethanol, (ii) are able to catabolize external ethanol, and (iii) that this catabolism affects their tendency to oscillate, we suggest that the dependence of the oscillations on cell density is mediated through the concentration of ethanol in the medium.

International Journal of Molecular Sciences, 2009

The pattern of flux and concentration control coefficients in an integrated mitochondrial energet... more The pattern of flux and concentration control coefficients in an integrated mitochondrial energetics model is examined by applying a generalized matrix method of control analysis to calculate control coefficients, as well as response coefficients The computational model of Cortassa et al. encompasses oxidative phosphorylation, the TCA cycle, and Ca 2+ dynamics. Control of ATP synthesis, TCA cycle, and ANT fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP/ADP production and transport, as well as by Ca 2+ dynamics. The calculation also analyzed the control of the concentrations of key regulatory ions and metabolites (Ca 2+ , NADH, ADP). The approach we have used demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

Mitochondrial Bioenergetics, 2011

The advent of techniques with the ability to scan massive changes in cellular makeup (genomics, p... more The advent of techniques with the ability to scan massive changes in cellular makeup (genomics, proteomics, etc.) has revealed the compelling need for analytical methods to interpret and make sense of those changes. Computational models built on sound physico-chemical mechanistic basis are unavoidable at the time of integrating, interpreting, and simulating high-throughput experimental data. Another powerful role of computational models is predicting new behavior provided they are adequately validated. Mitochondrial energy transduction has been traditionally studied with thermodynamic models. More recently, kinetic or thermo-kinetic models have been proposed, leading the path toward an understanding of the control and regulation of mitochondrial energy metabolism and its interaction with cytoplasmic and other compartments. In this work, we outline the methods, step-by-step, that should be followed to build a computational model of mitochondrial energetics in isolation or integrated to a network of cellular processes. Depending on the question addressed by the modeler, the methodology explained herein can be applied with different levels of detail, from the mitochondrial energy producing machinery in a network of cellular processes to the dynamics of a single enzyme during its catalytic cycle.

Circulation Research, 2006

Mitochondrial Ca 2+ ([Ca 2+ ] m ) regulates oxidative phosphorylation and thus contributes to ene... more Mitochondrial Ca 2+ ([Ca 2+ ] m ) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca 2+ via the Ca 2+ uniporter (MCU) and extrude it through the mitochondrial Na + /Ca 2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca 2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca 2+ ([Ca 2+ ] c ) transients. Furthermore, although mitochondrial Ca 2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na + ([Na + ] i ) affects mitochondrial Ca 2+ uptake and bioenergetics. To monitor [Ca 2+ ] m , mitochondria of guinea pig cardiac myocytes were loaded with rhod-2–acetoxymethyl ester (rhod-2 AM), and [Ca 2+ ] c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca 2+ ] c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca 2+ ] m transients, whose amplitude (Δ) correlated linearly ...

Circulation Research, 2007

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pu... more Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca 2+ . A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca 2+ pump and the ryanodine receptor 2, leading to increased Ca 2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca 2+ -release channels and accelerates Ca 2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca 2+ transport. Contraction improves with...

Biophysical Journal, 2012

Biophysical Journal, 2012

The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO... more The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) does not resolve the last six residues of the C-terminus of subunit I. In the higher resolution 2-subunit structure (PDB: 2GSM), ten more residues in the C-terminus are missing, suggesting flexibility of this region, which is also implied by deuterium exchange and computational analysis. To determine the functional significance of the C-terminus, three mutants of RsCcO were created, CID16 , CID6 and E552A. The 16-residue deletion mutant, CID16 (C-terminus = T550), showed suicide inactivation and lower steady state activity compared to wild type RsCcO. In the purified, reconstituted CID16, proton pumping is greatly impaired, though its respiratory control is good. SDS-PAGE indicates that subunit III is partially lost. The shorter deletion mutant, CID6 (C-terminus = W560), had normal activity and no suicide inactivation, suggesting that loss of activity in CID16 could be due to the importance of some residue(s) in the 551-560 region. A conserved glutamate, E552, was a likely candidate, and in fact the E552A mutant had normal activity but diminished proton-pumping. Crystal structures of CID16 and CID6 were obtained at 2.1 Å and 2.5 Å respectively. The CID16 structure shows a rearrangement of the new C-terminus (T550) to interact directly with H26, but the ordered waters above D132 in the D-pathway were unaltered. The CID6 structure appeared WT. A role for E552 in proton pumping is implied, and of the 551 to 560 region in stabilizing the association of subunit I and III. (NIH GM26916).

Biophysical Journal, 2012

The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO... more The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) does not resolve the last six residues of the C-terminus of subunit I. In the higher resolution 2-subunit structure (PDB: 2GSM), ten more residues in the C-terminus are missing, suggesting flexibility of this region, which is also implied by deuterium exchange and computational analysis. To determine the functional significance of the C-terminus, three mutants of RsCcO were created, CID16 , CID6 and E552A. The 16-residue deletion mutant, CID16 (C-terminus = T550), showed suicide inactivation and lower steady state activity compared to wild type RsCcO. In the purified, reconstituted CID16, proton pumping is greatly impaired, though its respiratory control is good. SDS-PAGE indicates that subunit III is partially lost. The shorter deletion mutant, CID6 (C-terminus = W560), had normal activity and no suicide inactivation, suggesting that loss of activity in CID16 could be due to the importance of some residue(s) in the 551-560 region. A conserved glutamate, E552, was a likely candidate, and in fact the E552A mutant had normal activity but diminished proton-pumping. Crystal structures of CID16 and CID6 were obtained at 2.1 Å and 2.5 Å respectively. The CID16 structure shows a rearrangement of the new C-terminus (T550) to interact directly with H26, but the ordered waters above D132 in the D-pathway were unaltered. The CID6 structure appeared WT. A role for E552 in proton pumping is implied, and of the 551 to 560 region in stabilizing the association of subunit I and III. (NIH GM26916).

Biophysical Journal, 2013

Type 2 diabetes mellitus (T2DM) is characterized by obesity, hyperglycemia and insulin resistance... more Type 2 diabetes mellitus (T2DM) is characterized by obesity, hyperglycemia and insulin resistance; these traits are recapitulated by the Zucker Diabetic Fatty (ZDF) rat model. Although fatty acid oxidation is known to be augmented in the heart, it is unclear how substrate fuel selection and redox behavior affect optimization of cardiac work output in T2DM.

Metabolic remodelling of glucose, fatty acid and redox pathways in the heart of type 2 diabetic mice

The Journal of Physiology, Apr 1, 2020

Hearts from type 2 diabetic animals display perturbations in excitation–contraction coupling, imp... more Hearts from type 2 diabetic animals display perturbations in excitation–contraction coupling, impairing myocyte contractility and delaying relaxation, along with altered substrate consumption patterns. Under high glucose and β‐adrenergic stimulation conditions, palmitate can, at least in part, offset left ventricle (LV) dysfunction in hearts from diabetic mice, improving contractility and relaxation while restoring coronary perfusion pressure. Fluxome calculations of central catabolism in diabetic hearts show that, in the presence of palmitate, there is a metabolic remodelling involving tricarboxylic acid cycle, polyol and pentose phosphate pathways, leading to improved redox balance in cytoplasmic and mitochondrial compartments. Under high glucose and increased energy demand, the metabolic/fluxomic redirection leading to restored redox balance imparted by palmitate helps explain maintained LV function and may contribute to designing novel therapeutic approaches to prevent cardiac dysfunction in diabetic patients.

To understand the control and regulation of mitochondrial energy metabolism a generalized matrix ... more To understand the control and regulation of mitochondrial energy metabolism a generalized matrix method of Metabolic Control Analysis has been applied to a computational model of mitochondrial energetics. The computational model of Cortassa et al. (2003) encompasses oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and ion dynamics across the inner mitochondrial membrane. Control of respiration and ATP synthesis fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP synthesis and ATP/ADP transport, as well as by Ca 2+ dynamics. The analysis of flux control coefficients and response coefficients has led to the notion of control by diffuse loops, that points to the regulatory interactions exerted by processes that are mechanistically only indirectly related with each other. The approach we have utilized demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

A Remarkable Adaptive Paradigm Of Heart Performance And Protection Emerges In Response To The Constitutive Challenge Of Marked Cardiac-Specific Overexpression Of Adenylyl Cyclase Type 8

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt ... more Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload (∼30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90α, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide arr...

Function, 2021

ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be ... more ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+: H+ stoichiometry. The excellent agreement between the functional data obtai...

Journal of Cell Science, 1991

The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investig... more The control exerted in vivo by mitochondrial functions on the dynamics of glycolysis was investigated in starved yeast cells that were metabolizing glucose semianaerobically. Glycolytic oscillations were triggered after a pulse of glucose by inhibition of mitochondrial respiration with KCN, myxothiazol and antimycin A or in mutants in the bel complex (ubiquinol:cytochrome c reductase) that were largely deficient in respiratory capacity. Inhibition of the adenine nucleotide translocator by preincubation with bongkrekic acid also triggered a train of damped sinusoidal oscillations after glucose addition. The oscillations consisted of cycles of reduction and oxidation of the intracellular pool of nicotinamide nucleotides with periods of 45 s to 1 min and amplitudes of 0.8 mM or lower. Preincubation with the uncoupler carbonyl cyamide p-(trifluoromethoxy)phenylhydrazone (FCCP) annihilated cyanide-induced oscillations of NAD(P)H. Evidence for de-energization of mitochondrial membranes in...

Frontiers in Physiology, 2020

Mitochondrial criticality describes a state in which the mitochondrial cardiac network under inte... more Mitochondrial criticality describes a state in which the mitochondrial cardiac network under intense oxidative stress becomes very sensitive to small perturbations, leading from local to cell-wide depolarization and synchronized oscillations that may escalate to the myocardial syncytium generating arrhythmias. Herein, we describe the occurrence of mitochondrial criticality in the chronic setting of a metabolic disorder, type 1 diabetes (T1DM), using a streptozotocin (STZ)-treated guinea pig (GP) animal model. Using wavelet analysis of mitochondrial networks from two-photon microscopy imaging of cardiac myocytes loaded with a fluorescent probe of the mitochondrial membrane potential, we show that cardiomyocytes from T1DM GPs are closer to criticality, making them more vulnerable to cell-wide mitochondrial oscillations as can be judged by the latency period to trigger oscillations after a laser flash perturbation, and their propensity to oscillate. Insulin treatment of T1DM GPs rescued cardiac myocytes to sham control levels of susceptibility, a protective condition that could also be attained with interventions leading to improvement of the cellular redox environment such as preincubation of diabetic cardiac myocytes with the lipid palmitate or a cell-permeable form of glutathione, in the presence of glucose.

Cell metabolism, Jan 6, 2018

The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD, i... more The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD, is elusive. Here, we report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Untargeted metabolite profiling of the liver and metabolic flux analysis of liver-derived cells revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Targeted NAD metabolome analysis in liver revealed depressed expression of NAM salvage in NAM-treated mice, an effect counteracted by higher expression of de novo NAD biosynthetic enzymes. Although neither hepatic NADnor NADPwas boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in ...

PLOS Computational Biology, 2017

Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknow... more Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze β-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO 2) and hydrogen peroxide (H 2 O 2) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H 2 O 2 emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of β-oxidation of long-chain fatty acids and main energyredox processes is able to simulate the relationship between VO 2 and H 2 O 2 emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H 2 O 2 emission as a function of VO 2. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply.

Processes, 2015

The advent of high throughput-omics has made the accumulation of comprehensive data sets possible... more The advent of high throughput-omics has made the accumulation of comprehensive data sets possible, consisting of changes in genes, transcripts, proteins and metabolites. Systems biology-inspired computational methods for translating metabolomics data into fluxomics provide a direct functional, dynamic readout of metabolic networks. When combined with appropriate experimental design, these methods deliver insightful knowledge about cellular function under diverse conditions. The use of computational models accounting for detailed kinetics and regulatory mechanisms allow us to unravel the control and regulatory properties of the fluxome under steady and time-dependent behaviors. This approach extends the analysis of complex systems from description to prediction, including control of complex dynamic behavior ranging from biological rhythms to catastrophic lethal arrhythmias. The powerful quantitative metabolomics-fluxomics approach will help our ability to engineer unicellular and multicellular organisms evolve from trial-and-error to a more predictable process, and from cells to organ and organisms.

2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2011

To understand the control and regulation of mitochondrial energy metabolism a generalized matrix ... more To understand the control and regulation of mitochondrial energy metabolism a generalized matrix method of Metabolic Control Analysis has been applied to a computational model of mitochondrial energetics. The computational model of Cortassa et al. (2003) encompasses oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and ion dynamics across the inner mitochondrial membrane. Control of respiration and ATP synthesis fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP synthesis and ATP/ADP transport, as well as by Ca 2+ dynamics. The analysis of flux control coefficients and response coefficients has led to the notion of control by diffuse loops, that points to the regulatory interactions exerted by processes that are mechanistically only indirectly related with each other. The approach we have utilized demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

Journal of General Microbiology, 1992

Cell synchrony was investigated during glycolytic oscillations in starved yeast cell suspensions ... more Cell synchrony was investigated during glycolytic oscillations in starved yeast cell suspensions at cell densities ranging from 2 x 106-5 x lo7 cells ml-l. Oscillations in NAD(P)H were triggered by inhibition of mitochondria1 respiration when intracellular NAD(P)H had reached a steady state after glucose addition. Before macroscopic damping of the oscillations, individual yeast cells oscillated in phase with the cell population. After oscillations had damped out macroscopically, a significant fraction of the cells still exhibited oscillatory dynamics, slightly out-ofphase. At cell concentrations higher than lo7 cells ml-l the dependence upon cell-density of (i) the damping of glycolytic oscillations and (ii) the amplitude per cell suggested that cell-to-cell interaction occurred. Most importantly, at cell densities exceeding lo7 cells ml-l the damping was much weaker. A combination of modelling studies and experimental analysis of the kinetics of damping of oscillations and their amplitude, with and without added ethanol, pyruvate or acetaldehyde, suggested that the autonomous glycolytic oscillations of the yeast cells depend upon the balance between oxidative and reductive (ethanol catabolism) fluxes of NADH, which is affected by the extracellular concentration of ethanol. Based on the facts that cell (i) excrete ethanol, (ii) are able to catabolize external ethanol, and (iii) that this catabolism affects their tendency to oscillate, we suggest that the dependence of the oscillations on cell density is mediated through the concentration of ethanol in the medium.

International Journal of Molecular Sciences, 2009

The pattern of flux and concentration control coefficients in an integrated mitochondrial energet... more The pattern of flux and concentration control coefficients in an integrated mitochondrial energetics model is examined by applying a generalized matrix method of control analysis to calculate control coefficients, as well as response coefficients The computational model of Cortassa et al. encompasses oxidative phosphorylation, the TCA cycle, and Ca 2+ dynamics. Control of ATP synthesis, TCA cycle, and ANT fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP/ADP production and transport, as well as by Ca 2+ dynamics. The calculation also analyzed the control of the concentrations of key regulatory ions and metabolites (Ca 2+ , NADH, ADP). The approach we have used demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis.

Mitochondrial Bioenergetics, 2011

The advent of techniques with the ability to scan massive changes in cellular makeup (genomics, p... more The advent of techniques with the ability to scan massive changes in cellular makeup (genomics, proteomics, etc.) has revealed the compelling need for analytical methods to interpret and make sense of those changes. Computational models built on sound physico-chemical mechanistic basis are unavoidable at the time of integrating, interpreting, and simulating high-throughput experimental data. Another powerful role of computational models is predicting new behavior provided they are adequately validated. Mitochondrial energy transduction has been traditionally studied with thermodynamic models. More recently, kinetic or thermo-kinetic models have been proposed, leading the path toward an understanding of the control and regulation of mitochondrial energy metabolism and its interaction with cytoplasmic and other compartments. In this work, we outline the methods, step-by-step, that should be followed to build a computational model of mitochondrial energetics in isolation or integrated to a network of cellular processes. Depending on the question addressed by the modeler, the methodology explained herein can be applied with different levels of detail, from the mitochondrial energy producing machinery in a network of cellular processes to the dynamics of a single enzyme during its catalytic cycle.

Circulation Research, 2006

Mitochondrial Ca 2+ ([Ca 2+ ] m ) regulates oxidative phosphorylation and thus contributes to ene... more Mitochondrial Ca 2+ ([Ca 2+ ] m ) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca 2+ via the Ca 2+ uniporter (MCU) and extrude it through the mitochondrial Na + /Ca 2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca 2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca 2+ ([Ca 2+ ] c ) transients. Furthermore, although mitochondrial Ca 2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na + ([Na + ] i ) affects mitochondrial Ca 2+ uptake and bioenergetics. To monitor [Ca 2+ ] m , mitochondria of guinea pig cardiac myocytes were loaded with rhod-2–acetoxymethyl ester (rhod-2 AM), and [Ca 2+ ] c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca 2+ ] c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca 2+ ] m transients, whose amplitude (Δ) correlated linearly ...

Circulation Research, 2007

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pu... more Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca 2+ . A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca 2+ pump and the ryanodine receptor 2, leading to increased Ca 2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca 2+ -release channels and accelerates Ca 2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca 2+ transport. Contraction improves with...

Biophysical Journal, 2012

Biophysical Journal, 2012

The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO... more The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) does not resolve the last six residues of the C-terminus of subunit I. In the higher resolution 2-subunit structure (PDB: 2GSM), ten more residues in the C-terminus are missing, suggesting flexibility of this region, which is also implied by deuterium exchange and computational analysis. To determine the functional significance of the C-terminus, three mutants of RsCcO were created, CID16 , CID6 and E552A. The 16-residue deletion mutant, CID16 (C-terminus = T550), showed suicide inactivation and lower steady state activity compared to wild type RsCcO. In the purified, reconstituted CID16, proton pumping is greatly impaired, though its respiratory control is good. SDS-PAGE indicates that subunit III is partially lost. The shorter deletion mutant, CID6 (C-terminus = W560), had normal activity and no suicide inactivation, suggesting that loss of activity in CID16 could be due to the importance of some residue(s) in the 551-560 region. A conserved glutamate, E552, was a likely candidate, and in fact the E552A mutant had normal activity but diminished proton-pumping. Crystal structures of CID16 and CID6 were obtained at 2.1 Å and 2.5 Å respectively. The CID16 structure shows a rearrangement of the new C-terminus (T550) to interact directly with H26, but the ordered waters above D132 in the D-pathway were unaltered. The CID6 structure appeared WT. A role for E552 in proton pumping is implied, and of the 551 to 560 region in stabilizing the association of subunit I and III. (NIH GM26916).

Biophysical Journal, 2012

The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO... more The 4-subunit crystal structure (PDB:1M56) of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) does not resolve the last six residues of the C-terminus of subunit I. In the higher resolution 2-subunit structure (PDB: 2GSM), ten more residues in the C-terminus are missing, suggesting flexibility of this region, which is also implied by deuterium exchange and computational analysis. To determine the functional significance of the C-terminus, three mutants of RsCcO were created, CID16 , CID6 and E552A. The 16-residue deletion mutant, CID16 (C-terminus = T550), showed suicide inactivation and lower steady state activity compared to wild type RsCcO. In the purified, reconstituted CID16, proton pumping is greatly impaired, though its respiratory control is good. SDS-PAGE indicates that subunit III is partially lost. The shorter deletion mutant, CID6 (C-terminus = W560), had normal activity and no suicide inactivation, suggesting that loss of activity in CID16 could be due to the importance of some residue(s) in the 551-560 region. A conserved glutamate, E552, was a likely candidate, and in fact the E552A mutant had normal activity but diminished proton-pumping. Crystal structures of CID16 and CID6 were obtained at 2.1 Å and 2.5 Å respectively. The CID16 structure shows a rearrangement of the new C-terminus (T550) to interact directly with H26, but the ordered waters above D132 in the D-pathway were unaltered. The CID6 structure appeared WT. A role for E552 in proton pumping is implied, and of the 551 to 560 region in stabilizing the association of subunit I and III. (NIH GM26916).

Biophysical Journal, 2013

Type 2 diabetes mellitus (T2DM) is characterized by obesity, hyperglycemia and insulin resistance... more Type 2 diabetes mellitus (T2DM) is characterized by obesity, hyperglycemia and insulin resistance; these traits are recapitulated by the Zucker Diabetic Fatty (ZDF) rat model. Although fatty acid oxidation is known to be augmented in the heart, it is unclear how substrate fuel selection and redox behavior affect optimization of cardiac work output in T2DM.