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Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 μM ouabain 100 nM blebbistatin) or chronic myocardial Na i load (PLM 3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic ‘fingerprint’. Control (PLM WT ), transgenic (PLM 3SA ), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23 Na, 31 P, 13 C NMR followed by 1 H-NMR metabolomic profiling. Elevated Na i leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Na i overload or inhibition of Na/Ca mito may be a new approach to ameliorate metabolic dysregulation in heart failure. The failing heart is characterised by both alterations in mitochondrial metabolism and an elevation of cytosolic sodium. Here, the authors use 23 Na NMR and metabolic profiling to show these are related, and that elevation in intracellular Na reprograms cardiac substrate utilisation via effects on mitochondrial Na/Ca exchange.

The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, metabolomics and RNAseq of cardiac tissue from naked mole-rats was compared to other African mole-rat genera (Cape, Cape dune, Common, Natal, Mahali, Highveld and Damaraland mole-rats) and evolutionarily divergent mammals (Hottentot golden mole and C57/BL6 mouse). We identify metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1α is observed while downstream hypoxia responsive-genes are down-regulated, suggesting adaptation to low oxygen environments. Naked mole-rat hearts show reduced succinate levels during ischemia compared to C57/BL6 mouse and negligible tissue damage following ischemia-reperfusion injury. These evolutionary traits reflect adaptation to a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span.

Elevated intracellular sodium Na i adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Na i decreases Gibb’s free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH 2 , which are all reversed on lowering Na i to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Na i elevation. Elevated Na i plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure. Heart failure is characterised by a detrimental rise in the intracellular sodium concentration. Here the authors show that this reversibly reprogrammes energy metabolism in the heart making this a possible therapeutic target for the development of new drugs.

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out.

Chronic kidney disease (CKD) is characterised by progressive loss of kidney function and structural damage, which contributes to systemic complications, including cardiovascular dysfunction. Inter-organ metabolic interactions are increasingly recognised as important in the pathophysiology of CKD, but the extent to which systemic bioenergetic deficits contribute to cardiac dysfunction remains unclear. We investigated cardiac and systemic metabolic remodeling in two rat models of CKD with distinct aetiologies: glomerulosclerosis induced by partial nephrectomy and interstitial fibrosis induced by an adenine-rich diet. Despite differing renal pathology, both models exhibited comparable cardiac dysfunction, including impaired recovery following 25 min of ischaemia. 1 H NMR spectroscopy metabolomic analysis revealed that systemic metabolic alterations in skeletal muscle, liver, and kidney were more pronounced than those in the heart, indicating reduced systemic bioenergetic reserve. These findings were supported by data from CKD patients, in whom 31 P NMR spectroscopy of exercising skeletal muscle demonstrated impaired phosphocreatine recovery, consistent with diminished bioenergetic capacity and reduced force generation. These results suggest that systemic bioenergetic impairment contributes to CKD-associated cardiac dysfunction. Targeting systemic metabolic derangements may represent a novel strategy to improve cardiac outcomes in CKD.

Copper-64-Diacetyl-bis(N 4 -methylthiosemicarbazone) [ 64 Cu][Cu(ATSM)] is a hypoxia-targeting PET tracer with applications in oncology and cardiology. Upon entering a hypoxic cell, [ 64 Cu][Cu(II)(ATSM)] is reduced to a putative [ 64 Cu][Cu(I)(ATSM)] − species which dissociates to deposit radiocopper, thereby providing hypoxic contrast. This process may be dependent upon protonation arising from intracellular acidosis. Since acidosis is a hallmark of ischemic tissue and tumors, the hypoxia specificity of [ 64 Cu][Cu(ATSM)] may be confounded by changes in intracellular pH. We have therefore determined the influence of intracellular pH on [ 64 Cu][Cu(ATSM)] pharmacokinetics. Using isolated perfused rat hearts, acidosis was induced using an ammonium pre-pulse method, with and without hypoxic buffer perfusion. Cardiac [ 64 Cu][Cu(ATSM)] pharmacokinetics were determined using NaI detectors, with intracellular pH and cardiac energetics monitored in parallel by 31 P NMR. To distinguish direct acidotic effects on tracer pharmacokinetics from acidosis-induced hypocontractility, parallel studies used lidocaine perfusion to abolish cardiac contraction. Hypoxic myocardium trapped [ 64 Cu][Cu(ATSM)] despite no evidence of it being acidotic when characterised by 31 P NMR. Independent induction of tissue acidosis had no direct effect on [ 64 Cu][Cu(ATSM)] pharmacokinetics in either normoxic or hypoxic hearts, beyond decreasing cardiac oxygen consumption to alleviate hypoxia and decrease tracer retention, leading us to conclude that tissue acidosis does not mediate the hypoxia selectivity of [ 64 Cu][Cu(ATSM)].

By the time cardiotoxicity-associated cardiac dysfunction is detectable by echocardiography it is often beyond meaningful intervention. 99m Tc-sestamibi is used clinically to image cardiac perfusion by single photon emission computed tomography (SPECT) imaging, but as a lipophilic cation its distribution is also governed by mitochondrial membrane potential (ΔΨ m ). Correcting scans for variations in perfusion (using a ΔΨ m -independent perfusion tracer such as (bis(N-ethoxy-N-ethyldithiocarbamato)nitrido 99m Tc(V)) ( 99m Tc-NOET) could allow 99m Tc-sestamibi to be repurposed to specifically report on ΔΨ m as a readout of evolving cardiotoxicity. Isolated rat hearts were perfused within a γ-detection apparatus to characterize the pharmacokinetics of 99m Tc-sestamibi and 99m Tc-NOET in response to mitochondrial perturbation by hypoxia, ionophore (CCCP) or doxorubicin. All interventions induced 99m Tc-sestamibi washout; hypoxia from 24.9 ± 2.6% ID to 0.4 ± 6.2%, CCCP from 22.8 ± 2.5% ID to −3.5 ± 3.1%, and doxorubicin from 23.0 ± 2.2% ID to 17.8 ± 0.7, p < 0.05. Cardiac 99m Tc-NOET retention (34.0 ± 8.0% ID) was unaffected in all cases. Translating to an i n vivo rat model, 2 weeks after bolus doxorubicin injection, there was a dose-dependent loss of cardiac 99m Tc-sestamibi retention (from 2.3 ± 0.3 to 0.9 ± 0.2 ID/g with 10 mg/kg (p < 0.05)), while 99m Tc-NOET retention (0.93 ± 0.16 ID/g) was unaffected. 99m Tc-NOET therefore traps in myocardium independently of the mitochondrial perturbations that induce 99m Tc-sestamibi washout, demonstrating proof-of-concept for an imaging approach to detect evolving cardiotoxicity.

Background Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na + /K + -ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. Methods Live whole-cell measurements of intracellular sodium [Na + ] i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23 Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2 H NMR to monitor conversion of [6,6- 2 H 2 ] d -glucose to [ 2 H]-labelled l -lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na + ] i was titrated using isotonic buffers with varying [Na + ] and [K + ] and introducing an artificial Na + plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1 H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[ 18 F]fluoroglucose ( 18 F-FDG) positron emission tomography (PET). Results Intracellular [Na + ] i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na + ] i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na + ] i in MDA-MB-231 and 4T1 cells and a Na + -dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18 FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18 FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. Conclusions Glycolytic flux correlates with Na + -driven NKA activity in breast cancer cells, providing evidence for the ‘centrality of the [Na + ] i -NKA nexus’ in the mechanistic basis of the Warburg effect.

Carnitine and its acyl derivatives are essential for the transport of fatty acids from the cytosol into the mitochondrial matrix for β‐oxidation, which supplies the cell with energy. Altered transport and metabolism of carnitine are associated with multiple diseases and disorders, including heart disease, insulin resistance, and cancer. Fluorinated carnitine derivatives have the potential to measure aberrant carnitine metabolism in these disorders using 19 F‐NMR and mass spectrometry. Furthermore, by radiolabeling carnitines with fluorine‐18, altered carnitine utilisation may be visualised in vivo using positron emission tomography (PET) imaging. Here, the design and synthesis of a fluorinated carnitine derivative, fluoromethylcarnitine (FMC), and its radiolabelled equivalent, [ 18 F]fluoromethylcarnitine ([ 18 F]FMC), are described, and their ability to quantitatively measure carnitine transport and downstream metabolism in a variety of settings are shown, from simple cell models to living subjects. Finally, [ 18 F]FMC PET is used to visualise elevated carnitine utilisation in a xenograft model of non‐small cell lung cancer.

Inducible nitric oxide synthase (NOS2) produces high local concentrations of nitric oxide (NO), and its expression is associated with inflammation, cellular stress signals, and cellular transformation. Additionally, NOS2 expression results in aggressive cancer cell phenotypes and is correlated with poor outcomes in patients with breast cancer. DNA hypomethylation, especially of noncoding repeat elements, is an early event in carcinogenesis and is a common feature of cancer cells. In addition to altered gene expression, DNA hypomethylation results in genomic instability via retrotransposon activation. Here, we show that NOS2 expression and associated NO signaling results in substantial DNA hypomethylation in human cell lines by inducing the degradation of DNA (cytosine-5)–methyltransferase 1 (DNMT1) protein. Similarly, NOS2 expression levels were correlated with decreased DNA methylation in human breast tumors. NOS2 expression and NO signaling also resulted in long interspersed noncoding element 1 (LINE-1) retrotransposon hypomethylation, expression, and DNA damage. DNMT1 degradation was mediated by an NO/p38-MAPK/lysine acetyltransferase 5–dependent mechanism. Furthermore, we show that this mechanism is required for NO-mediated epithelial transformation. Therefore, we conclude that NOS2 and NO signaling results in DNA damage and malignant cellular transformation via an epigenetic mechanism.