Search

Aims/Hypothesis Current drugs used to treat metabolic diseases such as obesity and type 2 diabetes have limited efficacy in directly normalising muscle insulin action, while exercise is effective in enhancing this aspect of metabolic disease. We recently identified Scriptaid as a compound that can replicate aspects of the exercise adaptive response, through disruption of the class IIa HDAC corepressor complex. The Aim of this study was to determine the effect of Scriptaid on muscle insulin action in obesity. Materials and Methods Diet-induced obese mice were administered Scriptaid (1 mg/kg) via daily i.p. injection for four weeks. Whole body and skeletal muscle metabolic phenotyping of mice was performed in addition to echocardiography to assess cardiac morphology and function. Results Scriptaid treatment had no effect on body weight or composition, but did increase energy expenditure, supported by increased lipid oxidation, while food intake was also increased. Scriptaid enhanced the expression of oxidative genes and proteins, increased fatty acid oxidation and reduced triglycerides and diacylglycerides in skeletal muscle. Furthermore, ex vivo insulin-stimulated glucose uptake by skeletal muscle was enhanced. Surprisingly, heart weight was reduced in Scriptaid-treated mice and was associated with enhanced expression of genes involved in oxidative metabolism in the heart. Scriptaid also improved indices of both diastolic and systolic cardiac function. Conclusion These data show that pharmacological targeting of the class IIa HDACs corepressor complex with Scriptaid could be used to enhance muscle insulin action and cardiac function in obesity.

Reduced nitric oxide (NO) bioavailability plays a central role in the pathogenesis of myocardial ischemia-reperfusion injury (I-R), and reduced l-arginine transport via cationic amino acid transporter-1 is a key contributor to the reduced NO levels. Insulin can increase NO levels by increasing the transport of its substrate l-arginine but insulin alone exerts minimal cardiac protection in I-R. We hypothesized that combined insulin and l-arginine may provide cardioprotective effects in the setting of myocardial I-R. The effect of supplemental insulin, l-arginine and the combination was examined in cardiomyocytes exposed to hypoxia/reoxygenation and in isolated perfused mouse hearts undergoing ischemia/reperfusion. When compared to controls, cardiomyocytes treated upon reoxygenation with 1nM insulin+1mM l-arginine exhibited significant (all P

BACKGROUND AND PURPOSE Annexin-A1 (ANX-A1) is an endogenous, glucocorticoid-regulated anti-inflammatory protein. The N-terminal-derived peptide Ac-ANX-A12–26 preserves cardiomyocyte viability, but the impact of ANX-A1-peptides on cardiac contractility is unknown. We now test the hypothesis that ANX-A1 preserves post-ischaemic recovery of left ventricular (LV) function. EXPERIMENTAL APPROACH Ac-ANX-A12–26 was administered on reperfusion, to adult rat cardiomyocytes as well as hearts isolated from rats, wild-type mice and mice deficient in endogenous ANX-A1 (ANX-A1–/–). Myocardial viability and recovery of LV function were determined. KEY RESULTS Ischaemia–reperfusion markedly impaired both cardiomyocyte viability and recovery of LV function by 60%. Treatment with exogenous Ac-ANX-A12–26 at the onset of reperfusion prevented cardiomyocyte injury and significantly improved recovery of LV function, in both intact rat and wild-type mouse hearts. Ac-ANX-A12–26 cardioprotection was abolished by either formyl peptide receptor (FPR)-nonselective or FPR1-selective antagonists, Boc2 and cyclosporin H, but was relatively insensitive to the FPR2-selective antagonist QuinC7. ANX-A1-induced cardioprotection was associated with increased phosphorylation of the cell survival kinase Akt. ANX-A1−/− exaggerated impairment of post-ischaemic recovery of LV function, in addition to selective LV FPR1 down-regulation. CONCLUSIONS AND IMPLICATIONS These data represent the first evidence that ANX-A1 affects myocardial function. Our findings suggest ANX-A1 is an endogenous regulator of post-ischaemic recovery of LV function. Furthermore, the ANX-A1-derived peptide Ac-ANX-A12–26 on reperfusion rescues LV function, probably via activation of FPR1. ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of myocardial reperfusion injury.

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired l-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the l-arginine transport inhibitor l-lysine (10 μmol·kg−1·min−1; 30 min) and subsequent superimposition of l-arginine (100 μmol·kg−1·min−1; 30 min), the NO synthase inhibitor NG-nitro-l-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 μg·kg−1·min−1) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). l-Lysine tended to reduce medullary perfusion (−15 ± 7%; P = 0.07) and reduced medullary NO concentration (−9 ± 3%; P = 0.03) while subsequent superimposition of l-arginine reversed these effects of l-lysine in SD rats. In SHR, l-lysine and subsequent superimposition of l-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal l-arginine transport is impaired in SHR. Renal l-[3H]arginine transport was less in SHR compared with SD rats ( P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney.

The action of oxidatively modified low-density lipoprotein on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. However, the biochemical mechanism for such action is not clear. We have previously shown that arginine uptake and metabolism are major determinants of endothelial function in heart failure and hypertension. In the present study we therefore aimed to assess the effects of oxidized LDL, a major pro-atherogenic molecule, on endothelial l-arginine metabolism and its uptake. Endothelial cells were exposed to oxidized LDL or native LDL for 24h, and the resultant effects on (1) the intracellular content of arginine and its major metabolites including citrulline, N(G)-hydroxy-l-arginine, asymmetric dimethylarginine, symmetric dimethylarginine and ornithine, (2) [3H]-l-arginine uptake and, (3) the pattern of distribution of cationic amino acid transporter 1, the principal l-arginine transporter, by confocal microscopy. Oxidized LDL (100 microg/mL) reduced intracellular arginine and N(G)-hydroxy-l-arginine contents by 56 and 71% (P

Cardiac ischemia reperfusion leads to oxidative stress and poor physiological recovery. Selenium deficiency down-regulates thioredoxin reductase (Txnrd) and glutathione peroxidase (Gpx) activity, impairing recovery from ischemia-reperfusion. Furthermore, selenium supplementation has been shown to be cardioprotective and lessens oxidative stress in reperfused rat hearts. In this study we have investigated the role of selenium in the mRNA expression of these, and related antioxidant proteins, post ischemia-reperfusion. Male rats were fed varying doses of selenium for five weeks. Hearts were isolated and perfused using the Langendorff method with 22.5 min of global ischemia and 45 min reperfusion. RNA was extracted for quantitative real-time PCR analysis of glutathione peroxidase (Gpx)-1 and 4, glutathione reductase (Gsr), thioredoxin peroxidase-2 (Prdx2), thioredoxin (Txn) and thioredoxin reductase (Txnrd)-1 and 2 gene expression. Selenium deficiency produced significant reductions in Gpx-1, Gpx-4, Prdx2, Txnrd-1 and Txnrd-2 expression. Conversely, selenium supplementation of 1000 μg/kg significantly up-regulated Gpx-1, Gpx-4, Txn, Txnrd-1 and Txnrd-2 transcription. Our results show selenium modulates the cardiac mRNA expression of thioredoxin and glutathione related enzymes post ischemia-reperfusion, and impacts on tolerance to ischemia-reperfusion. (Mol Cell Biochem 270: 131–138, 2005)

Oxidative stress occurs when cellular levels of reactive oxygen species exceed anti-oxidant capabilities and has been implicated in the pathogenesis of pre-eclampsia. In this study we have examined the tissue levels of endogenous anti-oxidant proteins (superoxide dismutase, glutathione peroxidase, thioredoxin reductase and thioredoxin) and the level of lipid and protein oxidation in placental samples from normal and pre-eclamptic pregnancies. Pre-eclamptic tissue homogenates demonstrated significantly increased levels of lipid peroxidation (20.68 +/- 7.811 microM protein versus 5.33 +/- 4.03 microM/mg protein, P < 0.001) and a trended increase in protein carbonyl concentration (248.1 +/- 97.71 units/mg protein versus 209.7 +/- 82.6 U/mg protein) when compared to controls. The levels and activities of the anti-oxidant proteins superoxide dismutase (2.48 +/- 0.6 U/mg protein versus 2.02 +/- 0.51 U/mg protein, P

Activin A, a member of the transforming growth factor-β superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia-reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia-reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30 min of ischaemia and 2h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia-reperfusion were absent in TLR4(-/-) mice. Pre-treatment with follistatin significantly reduced ischaemia-reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3h of hypoxia and 2h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia-reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.

Aim: Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial L-arginine transporter, cationic amino acid transporter-1 (CAT1), can prevent oxidative stress-induced hypertension. Methods: Wild-type mice (WT; n = 9) and endothelial CAT1 overexpressing (CAT+ )m ice (n = 6) had telemetry probes implanted for the measurement of mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Minipumps were implanted for infusion of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETCA; 30 mg kg 1 day 1 ; 14 days) or its saline vehicle. Baseline levels of MAP, HR and locomotor activity were determined before and during chronic DETCA administration. Mice were then killed, and their plasma and kidneys collected for analysis of F2-isoprostane levels. Results: Basal MAP was less in CAT+ (92 2 mmHg; n = 6) than in WT (98 2 mmHg; n = 9; P< 0.001). During DETCA infusion, MAP was increased in WT (by 4.2 0.5%; P < 0.001) but not in CAT+, when compared to appropriate controls (PDETCA*genotype = 0.006). DETCA infusion increased total plasma F2-isoprostane levels (by 67 11%; P = 0.05) in WT but not in CAT+. Total renal F2-isoprostane levels were greater during DETCA infusion in WT (by 72%; P < 0.001), but not in CAT+, compared to appropriate controls. Conclusion: Augmented endothelial L-arginine transport attenuated the prohypertensive effects of systemic and renal oxidative stress, suggesting that manipulation of endothelial CAT1 may provide a new therapeutic approach for the treatment of cardiovascular disease associated with oxidative stress.

Objective: A number of factors contribute to diabetesassociated vascular dysfunction. In the present study, we tested whether exposure to advanced glycation endproducts (AGEs) impairs vascular reactivity independently of hyperglycemia and examined the potential mechanisms responsible for diabetes and AGE-associated vascular dysfunction. Methods: Vasodilator function was studied using infusion of exogenous AGEs into Sprague-Dawley rats as compared with control and streptozotocin-induced diabetic rats all followed for 16 weeks (nU10 per group). The level of arginine metabolites and expression of endothelial nitric oxide synthase (eNOS) and downstream mediators of nitric oxide-dependent signaling were examined. To further explore these mechanisms, cultured bovine aortic endothelial cells (BAECs) were exposed to AGEs. Results: Both diabetic and animals infused with AGEmodified rat serum albumin (AGE-RSA) had significantly impaired vasodilatory response to acetylcholine. Unlike diabetes-associated endothelial dysfunction, AGE infusion was not associated with changes in plasma arginine metabolites, asymmetric dimethyl-L-arginine levels or eNOS expression. However, expression of the downstream mediator cGMP-dependent protein kinase 1 (PKG-1) was significantly reduced by both AGE exposure and diabetes. AGEs also augmented hyperglycemia-associated depletion in endothelial nitric oxide production and eNOS protein expression in vitro, and the novel AGE inhibitor, alagebrium chloride, partly restored these parameters. Conclusion: We demonstrate that AGEs represent a potentially important cause of vascular dysfunction, linked to the induction of nitric oxide resistance. These findings also emphasize the deleterious and potentially additive effects of AGEs and hyperglycemia in diabetic vasculature.

Myocardial injury due to ischemia-reperfusion (I-R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I-R process induces a state of insufficient L-arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I-R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia-reoxygenation significantly decreased L-arginine uptake and NO production (42 ± 2% and 71 ± 4%, respectively, both P

The development of diabetic cardiomyopathy is a key contributor to heart failure and mortality in obesity and type 2 diabetes (T2D). Current therapeutic interventions for T2D have limited impact on the development of diabetic cardiomyopathy. Clearly, new therapies are urgently needed. A potential therapeutic target is protein kinase D (PKD), which is activated by metabolic insults and implicated in the regulation of cardiac metabolism, contractility and hypertrophy. We therefore hypothesised that PKD inhibition would enhance cardiac function in T2D mice. We first validated the obese and T2D db/db mouse as a model of early stage diabetic cardiomyopathy, which was characterised by both diastolic and systolic dysfunction, without overt alterations in left ventricular morphology. These functional characteristics were also associated with increased PKD2 phosphorylation in the fed state and a gene expression signature characteristic of PKD activation. Acute administration of the PKD inhibitor CID755673 to normal mice reduced both PKD1 and 2 phosphorylation in a time and dose-dependent manner. Chronic CID755673 administration to T2D db/db mice for two weeks reduced expression of the gene expression signature of PKD activation, enhanced indices of both diastolic and systolic left ventricular function and was associated with reduced heart weight. These alterations in cardiac function were independent of changes in glucose homeostasis, insulin action and body composition. These findings suggest that PKD inhibition could be an effective strategy to enhance heart function in obese and diabetic patients and provide an impetus for further mechanistic investigations into the role of PKD in diabetic cardiomyopathy.

Endothelial dysfunction is a hallmark of cardiovascular disease, and the l -arginine:NO pathway plays a critical role in determining endothelial function. Recent studies suggest that smoking, a well-recognized risk factor for vascular disease, may interfere with l -arginine and NO metabolism; however, this remains poorly characterized. Accordingly, we performed a series of complementary in vivo and in vitro studies to elucidate the mechanism by which cigarette smoke adversely affects endothelial function. In current smokers, plasma levels of asymmetrical dimethyl-arginine (ADMA) were 80% higher ( P =0.01) than nonsmokers, whereas citrulline (17%; P l -arginine (34%; P l -arginine (57%), and arginine (23%), while increasing ADMA (129%). CSE significantly inhibited (38%) arginine uptake in conjunction with a 34% reduction in expression of the arginine transporter, CAT1. In conjunction with these studies, CSE significantly reduced the activity of eNOS and NO production by endothelial cells, while stimulating the production of reactive oxygen species. In conclusion, cigarette smoke adversely affects the endothelial l -arginine NO synthase pathway, resulting in reducing NO production and elevated oxidative stress. In conjunction, exposure to cigarette smoke increases ADMA concentration, the latter being a risk factor for cardiovascular disease.

Epidemiological studies and in vitro analysis demonstrate correlations between selenium status and human pre-eclampsia (PET). Selenium is an essential component in the anti-oxidant proteins glutathione peroxidase and thioredoxin reductase, which are produced in lower amounts in pre-eclamptic placenta. This study examined the effect of modulating dietary selenium content in pregnant rats. Rats were fed diets containing no selenium, 239 microg/kg selenium or 1000 microg/kg selenium, four weeks prior to and following conception. Significant pregnancy-specific increases in systolic blood pressure (116.4 +/- 5.2 mmHg vs 108 +/- 6.8 mmHg vs 111.4 +/- 4.7 mmHg) and proteinuria (9.68 +/- 2.12 microg/ml vs 5.93 +/- 1.59 microg/ml vs 4.43 +/- 0.96 microg/ml) were demonstrated in animals fed a selenium free-diet when compared with normal or high selenium diets. Placental weight and pup number were not affected by selenium deprivation, however a significant decrease in the pup weight was evident. Selenium deprivation caused dose-dependent decreases in liver glutathione peroxidase (28.55 +/- 3.82 mmoles/min/mg vs 34.68 +/- 8.64 mmoles/min/mg) and thioredoxin reductase (2.37 +/- 1.25 U/mg vs 6.68 +/- 1.82 U/mg) activity, whereas superoxide dismutase activity remained constant. Placental activity of these enzymes also decreased leading to oxidative stress as measured by increased lipid peroxides (17.92 +/- 1.78 micromoles/mg vs 8.30 +/- 5.52 micromoles/mg) and protein carbonyls in tissue extracts from selenium-free animals. These results suggest that selenium deficiency in pregnant rats leads to symptoms similar to those seen in human PET and may provide an experimental model for studying this complex disease.

BackgroundImpaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury.Methods and resultsIn mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (pConclusionThese data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.