Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart.

In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation drives part of the excessive superoxide production implicated in the pathogenesis of heart failure. Pacing-induced heart failure was performed in eight chronically instrumented dogs. Seven normal dogs served as control. End-stage failure occurred after 28 +/- 1 days of pacing, when left ventricular end-diastolic pressure reached 25 mm Hg. In left ventricular tissue homogenates, spontaneous superoxide generation measured by lucigenin (5 microM) chemiluminescence was markedly increased in heart failure (1338 +/- 419 vs. 419 +/- 102 AU/mg protein, P < 0.05), as were NADPH levels (15.4 +/- 1.5 vs. 7.5 +/- 1.5 micromol/gww, P < 0.05). Superoxide production was further stimulated by the addition of NADPH. The NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and the NO synthase inhibitor L-NAME (1 mM) both significantly lowered superoxide generation in failing heart homogenates by 80% and 76%, respectively. G6PD was upregulated and its activity higher in heart failure compared to control (0.61 +/- 0.10 vs. 0.24 +/- 0.03 nmol/min/mg protein, P < 0.05), while superoxide production decreased to normal levels in the presence of the G6PD inhibitor 6-aminonicotinamide. We conclude that the activation of myocardial G6PD is a novel mechanism that enhances NADPH availability and fuels superoxide-generating enzymes in heart failure

Role of NAD(P)H Oxidase in Superoxide Generation and Endothelial Dysfunction in Goto-Kakizaki (GK) Rats as a Model of Nonobese NIDDM

Background: Cardiovascular disease is the leading cause of mortality in diabetics, and it has a complex etiology that operates on several levels. Endothelial dysfunction and increased generation of reactive oxygen species are believed to be an underlying cause of vascular dysfunction and coronary artery disease in diabetes. This impairment is likely the result of decreased bioavailability of nitric oxide (NO) within the vasculature. However, it is unclear whether hyperglycemia per se stimulates NADPH oxidase-derived superoxide generation in vascular tissue. Methods and Results: This study focused on whether NADPH oxidase-derived superoxide is elevated in vasculature tissue evoking endothelial/smooth muscle dysfunction in the hyperglycemic (16964 mg%) Goto-Kakizaki (GK) rat. By dihydroethidine fluorescence staining, we determined that aorta superoxide levels were significantly elevated in 9 month-old GK compared with age matched Wistar (GK; 19566%, Wistar; 10063.5%). Consistent with these findings, 10 26 mol/L acetylcholine-induced relaxation of the carotid artery was significantly reduced in GK rats compared with age matched Wistar (GK; 4167%, Wistar; 10065%) and measurements in the aorta showed a similar trend (p =.08). In contrast, relaxation to the NO donor SNAP was unaltered in GK compared to Wistar. Endothelial dysfunction was reversed by lowering of superoxide with apocynin, a specific Nox inhibitor. Conclusions: The major findings from this study are that chronic hyperglycemia induces significant vascular dysfunction i

Down-Regulation of Replication Factor C-40 (RFC40) Causes Chromosomal Missegregation in Neonatal and Hypertrophic Adult Rat Cardiac Myocytes

BACKGROUND: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied. METHODS: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12. RESULTS: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers. CONCLUSION: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy

Glucose-6-Phosphate Dehydrogenase Is a Regulator of Vascular Smooth Muscle Contraction

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 μM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca2+ independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 μM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 μM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca2+]i and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca2+]i, thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca2+ entry and CA contractions evoked by membrane depolarization. Antioxid. Redox Signal. 14, 543–558

Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction. The role of glucose-6-phosphate dehydrogenase-derived NADPH

Increased oxidative stress is a known cause of cardiac dysfunction in animals and patients with diabetes, but the sources of reactive oxygen species [e.g., superoxide anion (O2−)] and the mechanisms underlying O2− production in diabetic hearts are not clearly understood. Our aim was to determine whether NADPH oxidase (Nox) is a source of O2− and whether glucose-6-phosphate dehydrogenase (G6PD)-derived NADPH plays a role in augmenting O2− generation in diabetes. We assessed cardiac function, Nox and G6PD activities, NADPH levels, and the activities of antioxidant enzymes in heart homogenates from young (9–11 wk old) Zucker lean and obese (fa/fa) rats. We found that myocardial G6PD activity was significantly higher in fa/fa than in lean rats, whereas superoxide dismutase and glutathione peroxidase activities were decreased (P < 0.05). O2− levels were elevated (70–90%; P < 0.05) in the diabetic heart, and this elevation was blocked by the Nox inhibitor gp-91ds-tat (50 μM) or by the mitochondrial respiratory chain inhibitors antimycin (10 μM) and rotenone (50 μM). Inhibition of G6PD by 6-aminonicotinamide (5 mM) and dihydroepiandrosterone (100 μM) also reduced (P < 0.05) O2− production. Notably, the activities of Nox and G6PD in the fa/fa rat heart were inhibited by chelerythrine, a protein kinase C inhibitor. Although we detected no changes in stroke volume, cardiac output, or ejection fraction, left ventricular diameter was slightly increased during diastole and systole, and left ventricular posterior wall thickness was decreased during systole (P < 0.05) in Zucker fa/fa rats. Our findings suggest that in a model of severe hyperlipidema and hyperglycemia Nox-derived O2− generation in the myocardium is fueled by elevated levels of G6PD-derived NADPH. Similar mechanisms were found to activate O2− production and induce endothelial dysfunction in aorta. Thus G6PD may be a useful therapeutic target for treating the cardiovascular disease associated with type 2 diabetes, if second-generation drugs specifically reducing the activity of G6PD to near normal levels are developed