Adenylate kinase AK2 isoform integral in embryo and adult heart homeostasis.

Adenylate kinase2 (AK2) catalyzes trans-compartmental nucleotide exchange, but the functional implications of this mitochondrial intermembrane isoform is only partially understood. Here, transgenic AK2-/- null homozygosity was lethal early in embryo, indicating a mandatory role for intact AK2 in utero development. In the adult, conditional organ-specific ablation of AK2 precipitated abrupt heart failure with Krebs cycle and glycolytic metabolite buildup, suggesting a vital contribution to energy demanding cardiac performance. Depressed pump function recovered to pre-deletion levels overtime, suggestive of an adaptive response. Compensatory upregulation of phosphotransferase AK1, AK3, AK4 isozymes, creatine kinase isoforms, and hexokinase, along with remodeling of cell cycle/growth genes and mitochondrial ultrastructure supported organ rescue. Taken together, the requirement of AK2 in early embryonic stages, and the immediate collapse of heart performance in the AK2-deficient postnatal state underscore a primordial function of the AK2 isoform. Unsalvageable in embryo, loss of AK2 in the adult heart was recoverable, underscoring an AK2-integrated bioenergetics system with innate plasticity to maintain homeostasis on demand.

The Modulating Effects of Cardiac Resynchronization Therapy on Myocardial Metabolism in Heart Failure.

Heart failure (HF) is associated with changes in cardiac substrate utilization and energy metabolism, including a decline in high-energy phosphate content, mitochondrial dysfunction, and phosphotransfer enzyme deficiency. A shift toward glucose metabolism was noted in the end stage of HF in animals, although HF in humans may not be associated with a shift toward predominant glucose utilization. Deficiencies of micronutrients are well-established causes of cardiomyopathy. Correction of these deficits can improve heart function. The genes governing the energy metabolism were predominantly underexpressed in nonischemic cardiomyopathy and hypertrophic cardiomyopathy but were overexpressed in ischemic cardiomyopathy. Cardiac resynchronization therapy (CRT) has been proven to increase cardiac efficiency without increasing myocardial oxygen consumption. Altered myocardial metabolism is normalized by CRT to improve ventricular function.

Cardiac resynchronization therapy induces adaptive metabolic transitions in the metabolomic profile of heart failure.

BACKGROUND: Heart failure (HF) is associated with ventricular dyssynchrony and energetic inefficiency, which can be alleviated by cardiac resynchronization therapy (CRT). The aim of this study was to determine the metabolomic signature in HF and its prognostic value regarding the response to CRT. METHODS AND RESULTS: This prospective study consisted of 24 patients undergoing CRT for advanced HF and 10 control patients who underwent catheter ablation for supraventricular arrhythmia but not CRT. Blood samples were collected before and 3 months after CRT. Metabolomic profiling of plasma samples was performed with the use of gas chromatography-mass spectrometry and nuclear magnetic resonance. The plasma metabolomic profile was altered in the HF patients, with a distinct panel of metabolites, including Krebs cycle and lipid, amino acid, and nucleotide metabolism. CRT improved the metabolomic profile. The succinate-glutamate ratio, an index of Krebs cycle activity, improved from 0.58 ± 0.13 to 2.84 ± 0.60 (P < .05). The glucose-palmitate ratio, an indicator of the balance between glycolytic and fatty acid metabolism, increased from 0.96 ± 0.05 to 1.54 ± 0.09 (P < .01). Compared with nonresponders to CRT, responders had a distinct baseline plasma metabolomic profile, including higher isoleucine, phenylalanine, leucine, glucose, and valine levels and lower glutamate levels at baseline (P < .05). CONCLUSIONS: CRT improves the plasma metabolomic profile of HF patients, indicating harmonization of myocardial energy substrate metabolism. CRT responders may have a favorable metabolomic profile as a potential biomarker for predicting CRT outcome.

Pyruvate dehydrogenase kinase regulates vascular inflammation in atherosclerosis and increases cardiovascular risk.

AIMS: Recent studies have revealed a close connection between cellular metabolism and the chronic inflammatory process of atherosclerosis. While the link between systemic metabolism and atherosclerosis is well established, the implications of altered metabolism in the artery wall are less understood. Pyruvate dehydrogenase kinase (PDK)-dependent inhibition of pyruvate dehydrogenase (PDH) has been identified as a major metabolic step regulating inflammation. Whether the PDK/PDH axis plays a role in vascular inflammation and atherosclerotic cardiovascular disease remains unclear. METHODS AND RESULTS: Gene profiling of human atherosclerotic plaques revealed a strong correlation between PDK1 and PDK4 transcript levels and the expression of pro-inflammatory and destabilizing genes. Remarkably, the PDK1 and PDK4 expression correlated with a more vulnerable plaque phenotype, and PDK1 expression was found to predict future major adverse cardiovascular events. Using the small-molecule PDK inhibitor dichloroacetate (DCA) that restores arterial PDH activity, we demonstrated that the PDK/PDH axis is a major immunometabolic pathway, regulating immune cell polarization, plaque development, and fibrous cap formation in Apoe-/- mice. Surprisingly, we discovered that DCA regulates succinate release and mitigates its GPR91-dependent signals promoting NLRP3 inflammasome activation and IL-1ß secretion by macrophages in the plaque. CONCLUSIONS: We have demonstrated for the first time that the PDK/PDH axis is associated with vascular inflammation in humans and particularly that the PDK1 isozyme is associated with more severe disease and could predict secondary cardiovascular events. Moreover, we demonstrate that targeting the PDK/PDH axis with DCA skews the immune system, inhibits vascular inflammation and atherogenesis, and promotes plaque stability features in Apoe-/- mice. These results point toward a promising treatment to combat atherosclerosis.

Compartmentation of membrane processes and nucleotide dynamics in diffusion-restricted cardiac cell microenvironment.

Orchestrated excitation-contraction coupling in heart muscle requires adequate spatial arrangement of systems responsible for ion movement and metabolite turnover. Co-localization of regulatory and transporting proteins into macromolecular complexes within an environment of microanatomical cell components raises intracellular diffusion barriers that hamper the mobility of metabolites and signaling molecules. Compared to substrate diffusion in the cytosol, diffusional restrictions underneath the sarcolemma are much larger and could impede ion and nucleotide movement by a factor of 10(3)-10(5). Diffusion barriers thus seclude metabolites within the submembrane space enabling rapid and vectorial effector targeting, yet hinder energy supply from the bulk cytosolic space implicating the necessity for a shunting transfer mechanism. Here, we address principles of membrane protein compartmentation, phosphotransfer enzyme-facilitated interdomain energy transfer, and nucleotide signal dynamics at the subsarcolemma-cytosol interface. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Dynamic phosphometabolomic profiling of human tissues and transgenic models by 18O-assisted ³¹P NMR and mass spectrometry.

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope (18)O-assisted (31)P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The (18)O labeling procedure is based on the incorporation of one (18)O into P(i) from [(18)O]H(2)O with each act of ATP hydrolysis and the distribution of (18)O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded (18)O-labeling procedures has revealed significant differences in the dynamics of G-6-P[(18)O] (glycolysis), G-3-P[(18)O] (substrate shuttle), and G-1-P[(18)O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[(18)O], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1-/-), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[(18)O] and decreased G-1-P[(18)O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit-/--deficient hearts is characterized by a higher G-6-[(18)O]P turnover rate, G-6-P levels, glycolytic capacity, γ/ß-phosphoryl of GTP[(18)O] turnover, as well as ß-[(18)O]ATP and ß-[(18)O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus, (18)O-assisted gas chromatography-mass spectrometry and (31)P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.

Electron spray ionization mass spectrometry and 2D 31P NMR for monitoring 18O/16O isotope exchange and turnover rates of metabolic oligophosphates.

A new method was here developed for the determination of (18)O-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, ß-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of (18)O/(16)O exchange was validated with gas chromatography-mass spectrometry and 2D (31)P NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D (31)P NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, (18)O-labeling kinetics and turnover rates of α-, ß-, and γ-phosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system.

18O-assisted dynamic metabolomics for individualized diagnostics and treatment of human diseases.

Technological innovations and translation of basic discoveries to clinical practice drive advances in medicine. Today's innovative technologies enable comprehensive screening of the genome, transcriptome, proteome, and metabolome. The detailed knowledge, converged in the integrated "omics" (genomics, transcriptomics, proteomics, and metabolomics), holds an immense potential for understanding mechanism of diseases, facilitating their early diagnostics, selecting personalized therapeutic strategies, and assessing their effectiveness. Metabolomics is the newest "omics" approach aimed to analyze large metabolite pools. The next generation of metabolomic screening requires technologies for high throughput and robust monitoring of metabolite levels and their fluxes. In this regard, stable isotope 18O-based metabolite tagging technology expands quantitative measurements of metabolite levels and turnover rates to all metabolites that include water as a reactant, most notably phosphometabolites. The obtained profiles and turnover rates are sensitive indicators of energy and metabolic imbalances like the ones created by genetic deficiencies, myocardial ischemia, heart failure, neurodegenerative disorders, etc. Here we describe and discuss briefly the potential use of dynamic phosphometabolomic platform for disease diagnostics currently under development at Mayo Clinic.

Stable Isotope Tracing Uncovers Reduced γ/ß-ATP Turnover and Metabolic Flux Through Mitochondrial-Linked Phosphotransfer Circuits in Aggressive Breast Cancer Cells.

Changes in dynamics of ATP γ- and ß-phosphoryl turnover and metabolic flux through phosphotransfer pathways in cancer cells are still unknown. Using 18O phosphometabolite tagging technology, we have discovered phosphotransfer dynamics in three breast cancer cell lines: MCF7 (non-aggressive), MDA-MB-231 (aggressive), and MCF10A (control). Contrary to high intracellular ATP levels, the 18O labeling method revealed a decreased γ- and ß-ATP turnover in both breast cancer cells, compared to control. Lower ß-ATP[18O] turnover indicates decreased adenylate kinase (AK) flux. Aggressive cancer cells had also reduced fluxes through hexokinase (HK) G-6-P[18O], creatine kinase (CK) [CrP[18O], and mitochondrial G-3-P[18O] substrate shuttle. Decreased CK metabolic flux was linked to the downregulation of mitochondrial MTCK1A in breast cancer cells. Despite the decreased overall phosphoryl flux, overexpression of HK2, AK2, and AK6 isoforms within cell compartments could promote aggressive breast cancer growth.

Rearrangement of energetic and substrate utilization networks compensate for chronic myocardial creatine kinase deficiency.

Plasticity of the cellular bioenergetic system is fundamental to every organ function, stress adaptation and disease tolerance. Here, remodelling of phosphotransfer and substrate utilization networks in response to chronic creatine kinase (CK) deficiency, a hallmark of cardiovascular disease, has been revealed in transgenic mouse models lacking either cytosolic M-CK (M-CK(-/-)) or both M-CK and sarcomeric mitochondrial CK (M-CK/ScCKmit(-/-)) isoforms. The dynamic metabolomic signatures of these adaptations have also been defined. Tracking perturbations in metabolic dynamics with (18)O and (13)C isotopes and (31)P NMR and mass spectrometry demonstrate that hearts lacking M-CK have lower phosphocreatine (PCr) turnover but increased glucose-6-phosphate (G-6-P) turnover, glucose utilization and inorganic phosphate compartmentation with normal ATP γ-phosphoryl dynamics. Hearts lacking both M-CK and sarcomeric mitochondrial CK have diminished PCr turnover, total phosphotransfer capacity and intracellular energetic communication but increased dynamics of ß-phosphoryls of ADP/ATP, G-6-P and γ-/ß-phosphoryls of GTP, indicating redistribution of flux through adenylate kinase (AK), glycolytic and guanine nucleotide phosphotransfer circuits. Higher glycolytic and mitochondrial capacities and increased glucose tolerance contributed to metabolic resilience of M-CK/ScCKmit(-/-) mice. Multivariate analysis revealed unique metabolomic signatures for M-CK(-/-) and M-CK/ScCKmit(-/-) hearts suggesting that rearrangements in phosphotransfer and substrate utilization networks provide compensation for genetic CK deficiency. This new information highlights the significance of integrated CK-, AK-, guanine nucleotide- and glycolytic enzyme-catalysed phosphotransfer networks in supporting the adaptivity and robustness of the cellular energetic system.

(31)P NMR correlation maps of (18)O/ (16)O chemical shift isotopic effects for phosphometabolite labeling studies.

Intramolecular correlations among the (18)O-labels of metabolic oligophosphates, mapped by J-decoupled (31)P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the (18)O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of (18)O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological usefulness of the J-decoupled (31)P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally (18)O labeled in perfused mammalian hearts.

Gas chromatography-mass spectrometry based 18O stable isotope labeling of Krebs cycle intermediates.

New technologies permit determining metabolomic profiles of human diseases by fingerprinting metabolites levels. However, to fully understand metabolomic phenotypes, metabolite levels and turnover rates are necessary to know. Krebs cycle is the major hub of energy metabolism and cell signaling. Traditionally, 13C stable isotope labeled substrates were used to track the carbon turnover rates in Krebs cycle metabolites. In this study, for the first time we introduce H2[18O] based stable isotope marker that permit tracking oxygen exchange rates in separate segments of Krebs cycle. The chromatographic and non-chromatographic parameters were systematically tested on the effect of labeling ratio of Krebs cycle mediators to increase selectivity and sensitivity of the method. We have developed a rapid, precise, and robust GC-MS method for determining the percentage of 18O incorporation to Krebs cycle metabolites. The developed method was applied to track the cancer-induced shift in the Krebs cycle dynamics of Caco-2 cells as compared to the control FHC cells revealing Warburg effects in Caco-2 cells. We demonstrate that unique information could be obtained using this newly developed 18O-labeling analytical technology by following the oxygen exchange rates of Krebs cycle metabolites. Thus, 18O-labeling of Krebs cycle metabolites expands the arsenal of techniques for monitoring the dynamics of cellular metabolism. Moreover, the developed method will allow to apply the 18O-labeling technique to numerous other metabolic pathways where oxygen exchange with water takes place.

Partial inhibition of mitochondrial complex I ameliorates Alzheimer's disease pathology and cognition in APP/PS1 female mice.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

Glycolytic network restructuring integral to the energetics of embryonic stem cell cardiac differentiation.

Decoding of the bioenergetic signature underlying embryonic stem cell cardiac differentiation has revealed a mandatory transformation of the metabolic infrastructure with prominent mitochondrial network expansion and a distinctive switch from glycolysis to oxidative phosphorylation. Here, we demonstrate that despite reduction in total glycolytic capacity, stem cell cardiogenesis engages a significant transcriptome, proteome, as well as enzymatic and topological rearrangement in the proximal, medial, and distal modules of the glycolytic pathway. Glycolytic restructuring was manifested by a shift in hexokinase (Hk) isoforms from Hk-2 to cardiac Hk-1, with intracellular and intermyofibrillar localization mapping mitochondrial network arrangement. Moreover, upregulation of cardiac-specific enolase 3, phosphofructokinase, and phosphoglucomutase and a marked increase in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) phosphotransfer activity, along with apparent post-translational modifications of GAPDH and phosphoglycerate kinase, were all distinctive for derived cardiomyocytes compared to the embryonic stem cell source. Lactate dehydrogenase (LDH) isoforms evolved towards LDH-2 and LDH-3, containing higher proportions of heart-specific subunits, and pyruvate dehydrogenase isoforms rearranged between E1alpha and E1beta, transitions favorable for substrate oxidation in mitochondria. Concomitantly, transcript levels of fetal pyruvate kinase isoform M2, aldolase 3, and transketolase, which shunt the glycolytic with pentose phosphate pathways, were reduced. Collectively, changes in glycolytic pathway modules indicate active redeployment, which would facilitate connectivity of the expanding mitochondrial network with ATP utilization sites. Thus, the delineated developmental dynamics of the glycolytic phosphotransfer network is integral to the remodeling of cellular energetic infrastructure underlying stem cell cardiogenesis.

Adenylate Kinase and Metabolic Signaling in Cancer Cells.

A hallmark of cancer cells is the ability to rewire their bioenergetics and metabolic signaling circuits to fuel their uncontrolled proliferation and metastasis. Adenylate kinase (AK) is the critical enzyme in the metabolic monitoring of cellular adenine nucleotide homeostasis. It also directs AK→ AMP→ AMPK signaling controlling cell cycle and proliferation, and ATP energy transfer from mitochondria to distribute energy among cellular processes. The significance of AK isoform network in the regulation of a variety of cellular processes, which include cell differentiation and motility, is rapidly growing. Adenylate kinase 2 (AK2) isoform, localized in intermembrane and intra-cristae space, is vital for mitochondria nucleotide exchange and ATP export. AK2 deficiency disrupts cell energetics, causes severe human diseases, and is embryonically lethal in mice, signifying the importance of catalyzed phosphotransfer in cellular energetics. Suppression of AK phosphotransfer and AMP generation in cancer cells and consequently signaling through AMPK could be an important factor in the initiation of cancerous transformation, unleashing uncontrolled cell cycle and growth. Evidence also builds up that shift in AK isoforms is used later by cancer cells for rewiring energy metabolism to support their high proliferation activity and tumor progression. As cell motility is an energy-consuming process, positioning of AK isoforms to increased energy consumption sites could be an essential factor to incline cancer cells to metastases. In this review, we summarize recent advances in studies of the significance of AK isoforms involved in cancer cell metabolism, metabolic signaling, metastatic potential, and a therapeutic target.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Adenylate kinase and AMP signaling networks: metabolic monitoring, signal communication and body energy sensing.

Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.

Guided stem cell cardiopoiesis: discovery and translation.

Over 1000 patients have participated worldwide in clinical trials exploring the therapeutic value of bone marrow-derived cells in ischemic heart disease. Meta-analysis evaluation of this global effort indicates that adult stem cell therapy is in general safe, but yields a rather modest level of improvement in cardiac function and structural remodeling in the setting of acute myocardial infarction or chronic heart failure. Although promising, the potential of translating adult stem cell-based therapy from bench to bedside has yet to be fully realized. Inter-trial and inter-patient variability contribute to disparity in the regenerative potential of transplanted stem cells with unpredictable efficacy on follow-up. Strategies that mimic the natural embryonic program for uniform recruitment of cardiogenic progenitors from adult sources are currently tested to secure consistent outcome. Guided cardiopoiesis has been implemented with mesenchymal stem cells obtained from bone marrow of healthy volunteers, using a cocktail of secreted proteins that recapitulate components of the endodermal secretome critical for cardiogenic induction of embryonic mesoderm. With appropriate validation of this newly derived cardiopoietic phenotype, the next generation of trials should achieve demonstrable benefit across patient populations.