Guanidinoacetate
-methyltransferase (GAMT) is the second essential enzyme in creatine (Cr) biosynthesis. Short-term Cr deficiency is metabolically well tolerated as GAMT
mice exhibit normal exercise ...capacity and response to ischemic heart failure. However, we hypothesized long-term consequences of Cr deficiency and/or accumulation of the Cr precursor guanidinoacetate (GA).
Cardiac function and metabolic profile were studied in GAMT
mice >1 year.
LV catheterization revealed lower heart rate and developed pressure in aging GAMT
but normal lung weight and survival versus age-matched controls. Electron microscopy indicated reduced mitochondrial volume density in GAMT
hearts (
< 0.001), corroborated by lower mtDNA copy number (
< 0.004), and citrate synthase activity (
< 0.05), however, without impaired mitochondrial respiration. Furthermore, myocardial energy stores and key ATP homeostatic enzymes were barely altered, while pathology was unrelated to oxidative stress since superoxide production and protein carbonylation were unaffected. Gene expression of PGC-1α was 2.5-fold higher in GAMT
hearts while downstream genes were not activated, implicating a dysfunction in mitochondrial biogenesis signaling. This was normalized by 10 days of dietary Cr supplementation, as were all
functional parameters, however, it was not possible to differentiate whether relief from Cr deficiency or GA toxicity was causative.
Long-term Cr deficiency in GAMT
mice reduces mitochondrial volume without affecting respiratory function, most likely due to impaired biogenesis. This is associated with hemodynamic changes without evidence of heart failure, which may represent an acceptable functional compromise in return for reduced energy demand in aging mice.
Mitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia-reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a ...disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the "deactive" state, usually formed only after prolonged inactivity. Despite its tendency to adopt the "deactive" state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.
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
load (PLM
mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLM
), transgenic (PLM
), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by
Na,
P,
C NMR followed by
H-NMR metabolomic profiling. Elevated Na
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
overload or inhibition of Na/Ca
may be a new approach to ameliorate metabolic dysregulation in heart failure.
Multiple studies suggest creatine mediates anti-oxidant activity in addition to its established role in cellular energy metabolism. The functional significance for the heart has yet to be ...established, but antioxidant activity could contribute to the cardioprotective effect of creatine in ischaemia/reperfusion injury.
To determine whether intracellular creatine levels influence responses to acute reactive oxygen species (ROS) exposure in the intact beating heart. We hypothesised that mice with elevated creatine due to over-expression of the creatine transporter (CrT-OE) would be relatively protected, while mice with creatine-deficiency (GAMT KO) would fare worse.
CrT-OE mice were pre-selected for creatine levels 20-100% above wild-type using in vivo (1)H-MRS. Hearts were perfused in isovolumic Langendorff mode and cardiac function monitored throughout. After 20 min equilibration, hearts were perfused with either H2O2 0.5 µM (30 min), or the anti-neoplastic drug doxorubicin 15 µM (100 min). Protein carbonylation, creatine kinase isoenzyme activities and phospho-PKCδ expression were quantified in perfused hearts as markers of oxidative damage and apoptotic signalling. Wild-type hearts responded to ROS challenge with a profound decline in contractile function that was ameliorated by co-administration of catalase or dexrazoxane as positive controls. In contrast, the functional deterioration in CrT-OE and GAMT KO hearts was indistinguishable from wild-type controls, as was the extent of oxidative damage and apoptosis. Exogenous creatine supplementation also failed to protect hearts from doxorubicin-induced dysfunction.
Intracellular creatine levels do not influence the response to acute ROS challenge in the intact beating heart, arguing against creatine exerting (patho-)physiologically relevant anti-oxidant activity.
Elevated intracellular Na (Nai) and metabolic impairment are interrelated pathophysiological features of the failing heart (HF). There have been a number of studies showing that myocardial sodium ...elevation subtly affects mitochondrial function. During contraction, mitochondrial calcium (Camito) stimulates a variety of TCA cycle enzymes, thereby providing reducing equivalents to maintain ATP supply. Nai elevation has been shown to impact Camito; however, whether metabolic remodelling in HF is caused by increased Nai has only been recently demonstrated. This novel insight may help to elucidate the contribution of metabolic remodelling in the pathophysiology of HF, the lack of efficacy of current HF therapies and a rationale for the development of future metabolism-targeting treatments. Here we review the relationship between Na pump inhibition, elevated Nai, and altered metabolic profile in the context of HF and their link to metabolic (in)flexibility and mitochondrial reprogramming.
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•Myocardial Na elevation is a hallmark of heart failure.•Na affects mitochondrial function.•Na elevation precedes myocardial metabolic remodelling.•Prevention of myocardial Nai elevation could alleviate metabolic remodelling in HF.
Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic ...tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.
Aims
To measure the activity of the key phosphotransfer enzymes creatine kinase (CK), adenylate kinase (AK), and glycolytic enzymes in two common mouse models of chronic heart failure.
Methods and ...results
C57BL/6 mice were subjected to transverse aortic constriction (TAC), myocardial infarction induced by coronary artery ligation (CAL), or sham operation. Activities of phosphotransfer enzymes CK, AK, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), 3‐phosphoglycerate kinase (PGK), and pyruvate kinase were assessed spectrophotometrically. Mice were characterized by echocardiography or magnetic resonance imaging 5‐ to 8‐week post‐surgery and selected for the presence of congestive heart failure. All mice had severe left ventricular hypertrophy, impaired systolic function and pulmonary congestion compared with sham controls. A significant decrease in myocardial CK and maximal CK reaction velocity was observed in both experimental models of heart failure. However, the activity of AK and its isoforms remained unchanged, despite a reduction in its protein expression. In contrast, the activities of glycolytic phosphotransfer mediators GAPDH and PGK were 19 and 12% higher in TAC, and 31 and 23% higher in CAL models, respectively.
Conclusion
Chronic heart failure in the mouse is characterized by impaired CK function, unaltered AK, and increased activity of glycolytic phosphotransfer enzymes. This pattern of altered phosphotransfer activity was observed independent of the heart failure aetiology.
Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are ...tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo.
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•We have developed a mitochondria-targeted redox cycler, MitoPQ.•MitoPQ selectively increases superoxide within mitochondria.•This development addresses an unmet need to generate mitochondrial superoxide.•MitoPQ is a useful tool in mitochondrial and redox biology.
ABSTRACT
The role of peroxisome proliferator‐activated receptor α (PPARα)‐mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 ...wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using 31P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using 3H labeling. Compared with a normoxic, chow‐fed control mouse heart, hypoxia decreased PPARa expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations (ATP) and thereby, ejection fractions. Ahigh‐fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high‐fat diet, with the result that ATP and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have occurred already in PPARa‐deficient (PPARa‐/‐) mouse hearts and sustained function in hypoxia despite an inability for further metabolic remodeling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodeling in hypoxia, but is prevented by dietary fat.—Cole, M. A., Abd Jamil, A. H., Heather, L. C., Murray, A. J., Sutton, E. R., Slingo, M., Sebag‐Montefiore, L., Tan, S. C., Aksentijević, D., Gildea, O. S., Stuckey, D. J., Yeoh, K. K., Carr, C. A., Evans, R. D., Aasum, E., Schofield, C. J., Ratcliffe, P. J., Neubauer, S., Robbins, P. A., Clarke, K. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury. FASEB J. 30, 2684‐2697 (2016). www.fasebj.org
Abstract
Aims
Succinate accumulates several-fold in the ischaemic heart and is then rapidly oxidized upon reperfusion, contributing to reactive oxygen species production by mitochondria. In ...addition, a significant amount of the accumulated succinate is released from the heart into the circulation at reperfusion, potentially activating the G-protein-coupled succinate receptor (SUCNR1). However, the factors that determine the proportion of succinate oxidation or release, and the mechanism of this release, are not known.
Methods and results
To address these questions, we assessed the fate of accumulated succinate upon reperfusion of anoxic cardiomyocytes, and of the ischaemic heart both ex vivo and in vivo. The release of accumulated succinate was selective and was enhanced by acidification of the intracellular milieu. Furthermore, pharmacological inhibition, or haploinsufficiency of the monocarboxylate transporter 1 (MCT1) significantly decreased succinate efflux from the reperfused heart.
Conclusion
Succinate release upon reperfusion of the ischaemic heart is mediated by MCT1 and is facilitated by the acidification of the myocardium during ischaemia. These findings will allow the signalling interaction between succinate released from reperfused ischaemic myocardium and SUCNR1 to be explored.
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