Dunja Aksentijevic

Fumarate Is Cardioprotective via Activation of the Nrf2 Antioxidant Pathway

Summary The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarates cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Living without creatine: unchanged exercise capacity and response to chronic myocardial infarction in creatine-deficient mice.

Rationale: Creatine is thought to be involved in the spatial and temporal buffering of ATP in energetic organs such as heart and skeletal muscle. Creatine depletion affects force generation during maximal stimulation, while reduced levels of myocardial creatine are a hallmark of the failing heart, leading to the widely held view that creatine is important at high workloads and under conditions of pathological stress. Objective: We therefore hypothesised that the consequences of creatine-deficiency in mice would be impaired running capacity, and exacerbation of heart failure following myocardial infarction. Methods and Results: Surprisingly, mice with whole-body creatine deficiency due to knockout of the biosynthetic enzyme (guanidinoacetate N-methyltransferase [GAMT]) voluntarily ran just as fast and as far as controls (>10 km/night) and performed the same level of work when tested to exhaustion on a treadmill. Furthermore, survival following myocardial infarction was not altered, nor was subsequent left ventricular (LV) remodelling and development of chronic heart failure exacerbated, as measured by 3D-echocardiography and invasive hemodynamics. These findings could not be accounted for by compensatory adaptations, with no differences detected between WT and GAMT−/− proteomes. Alternative phosphotransfer mechanisms were explored; adenylate kinase activity was unaltered, and although GAMT−/− hearts accumulated the creatine precursor guanidinoacetate, this had negligible energy-transfer activity, while mitochondria retained near normal function. Conclusions: Creatine-deficient mice show unaltered maximal exercise capacity and response to chronic myocardial infarction, and no obvious metabolic adaptations. Our results question the paradigm that creatine is essential for high workload and chronic stress responses in heart and skeletal muscle. # Novelty and Significance {#article-title-47}Rationale: Creatine is thought to be involved in the spatial and temporal buffering of ATP in energetic organs such as heart and skeletal muscle. Creatine depletion affects force generation during maximal stimulation, while reduced levels of myocardial creatine are a hallmark of the failing heart, leading to the widely held view that creatine is important at high workloads and under conditions of pathological stress. Objective: We therefore hypothesised that the consequences of creatine-deficiency in mice would be impaired running capacity, and exacerbation of heart failure following myocardial infarction. Methods and Results: Surprisingly, mice with whole-body creatine deficiency due to knockout of the biosynthetic enzyme (guanidinoacetate N-methyltransferase [GAMT]) voluntarily ran just as fast and as far as controls (>10 km/night) and performed the same level of work when tested to exhaustion on a treadmill. Furthermore, survival following myocardial infarction was not altered, nor was subsequent left ventricular (LV) remodelling and development of chronic heart failure exacerbated, as measured by 3D-echocardiography and invasive hemodynamics. These findings could not be accounted for by compensatory adaptations, with no differences detected between WT and GAMT−/− proteomes. Alternative phosphotransfer mechanisms were explored; adenylate kinase activity was unaltered, and although GAMT−/− hearts accumulated the creatine precursor guanidinoacetate, this had negligible energy-transfer activity, while mitochondria retained near normal function. Conclusions: Creatine-deficient mice show unaltered maximal exercise capacity and response to chronic myocardial infarction, and no obvious metabolic adaptations. Our results question the paradigm that creatine is essential for high workload and chronic stress responses in heart and skeletal muscle.

Moderate elevation of intracellular creatine by targeting the creatine transporter protects mice from acute myocardial infarction

Aims Increasing energy storage capacity by elevating creatine and phosphocreatine (PCr) levels to increase ATP availability is an attractive concept for protecting against ischaemia and heart failure. However, testing this hypothesis has not been possible since oral creatine supplementation is ineffectual at elevating myocardial creatine levels. We therefore used mice overexpressing creatine transporter in the heart (CrT-OE) to test for the first time whether elevated creatine is beneficial in clinically relevant disease models of heart failure and ischaemia/reperfusion (I/R) injury. Methods and results CrT-OE mice were selected for left ventricular (LV) creatine 20–100% above wild-type values and subjected to acute and chronic coronary artery ligation. Increasing myocardial creatine up to 100% was not detrimental even in ageing CrT-OE. In chronic heart failure, creatine elevation was neither beneficial nor detrimental, with no effect on survival, LV remodelling or dysfunction. However, CrT-OE hearts were protected against I/R injury in vivo in a dose-dependent manner (average 27% less myocardial necrosis) and exhibited greatly improved functional recovery following ex vivo I/R (59% of baseline vs. 29%). Mechanisms contributing to ischaemic protection in CrT-OE hearts include elevated PCr and glycogen levels and improved energy reserve. Furthermore, creatine loading in HL-1 cells did not alter antioxidant defences, but delayed mitochondrial permeability transition pore opening in response to oxidative stress, suggesting an additional mechanism to prevent reperfusion injury. Conclusion Elevation of myocardial creatine by 20–100% reduced myocardial stunning and I/R injury via pleiotropic mechanisms, suggesting CrT activation as a novel, potentially translatable target for cardiac protection from ischaemia.

Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat

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.

High-energy phosphotransfer in the failing mouse heart: role of adenylate kinase and glycolytic enzymes

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.

Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT-/- mouse hearts.

Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes.

Increased oxidative metabolism following hypoxia in the type 2 diabetic heart, despite normal hypoxia signalling and metabolic adaptation

Adaptation to hypoxia makes the heart more oxygen efficient, by metabolising more glucose. In contrast, type 2 diabetes makes the heart metabolise more fatty acids. Diabetes increases the chances of the heart being exposed to hypoxia, but whether the diabetic heart can adapt and respond is unknown. In this study we show that diabetic hearts retain the ability to adapt their metabolism in response to hypoxia, with functional hypoxia signalling pathways. However, the hypoxia‐induced changes in metabolism are additive to abnormal baseline metabolism, resulting in hypoxic diabetic hearts metabolising more fat and less glucose than controls. This stops the diabetic heart being able to recover its function when stressed. These results demonstrate that the diabetic heart retains metabolic flexibility to adapt to hypoxia, but is hindered by the baseline effects of the disease. This increases our understanding of how the diabetic heart is affected by hypoxia‐associated complications of the disease.

Insulin resistance and altered glucose transporter 4 expression in experimental uremia

Progressive ventricular hypertrophy can lead to the development of insulin resistance, a feature of both chronic kidney disease and heart failure. Here we induced uremia in adult male Sprague-Dawley rats using a remnant kidney model and studied the expression of glucose transporters. As expected, the reduction of nephron mass resulted in impaired renal function, cardiac hypertrophy, glucose intolerance, hyperinsulinemia, anemia, and hypertension. Insulin sensitivity was significantly reduced in the uremic animals as determined by oral glucose tolerance tests. After six weeks of uremia, at a point when cardiac hypertrophy had been established, left ventricle tissue had a marked increase in the expression of GLUT4 (insulin-dependent glucose transporter 4), consistent with hypertrophic remodeling, but not GLUT1 (insulin-independent glucose transporter 1). However, although uremic animals had systemic insulin resistance and glucose intolerance, there was no evidence of impaired GLUT4 translocation in the heart at 6 weeks of uremia, suggesting that other mechanisms may underpin insulin resistance in the uremic heart.

On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury

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

Myocardial creatine levels do not influence response to acute oxidative stress in isolated perfused heart

Background 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. Objectives 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. Methods and Results CrT-OE mice were pre-selected for creatine levels 20–100% above wild-type using in vivo 1H–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. Conclusions 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.