Alison C. Cave

Increased myocardial NADPH oxidase activity in human heart failure

OBJECTIVES This study was designed to investigate whether nicotinamide adenine dinucleotide 3-phosphate (reduced form) (NADPH) oxidase is expressed in the human heart and whether it contributes to reactive oxygen species (ROS) production in heart failure. BACKGROUND A phagocyte-type NADPH oxidase complex is a major source of ROS in the vasculature and is implicated in the pathophysiology of hypertension and atherosclerosis. An increase in myocardial oxidative stress due to excessive production of ROS may be involved in the pathophysiology of congestive heart failure. Recent studies have suggested an important role for myocardial NADPH oxidase in experimental models of cardiac disease. However, it is unknown whether NADPH oxidase is expressed in the human myocardium or if it has any role in human heart failure. METHODS Myocardium of explanted nonfailing (n = 9) and end-stage failing (n = 13) hearts was studied for the expression of NADPH oxidase subunits and oxidase activity. RESULTS The NADPH oxidase subunits p22(phox), gp91(phox), p67(phox), and p47(phox) were all expressed at messenger ribonucleic acid and protein level in cardiomyocytes of both nonfailing and failing hearts. NADPH oxidase activity was significantly increased in end-stage failing versus nonfailing myocardium (5.86 +/- 0.41 vs. 3.72 +/- 0.39 arbitrary units; p < 0.01). The overall level of oxidase subunit expression was unaltered in failing compared with nonfailing hearts. However, there was increased translocation of the regulatory subunit, p47(phox), to myocyte membranes in failing myocardium. CONCLUSIONS This is the first report of the presence of NADPH oxidase in human myocardium. The increase in NADPH oxidase activity in the failing heart may be important in the pathophysiology of cardiac dysfunction by contributing to increased oxidative stress.

Contrasting Roles of NADPH Oxidase Isoforms in Pressure-Overload Versus Angiotensin II–Induced Cardiac Hypertrophy

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)–induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox−/− mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II–induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox−/− mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox−/− and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox−/− mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload–induced LVH in both gp91phox−/− and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload.

Aldosterone mediates angiotensin II-induced interstitial cardiac fibrosis via a Nox2-containing NADPH oxidase

Angiotensin (ANG) II (AngII) and aldosterone contribute to the development of interstitial cardiac fibrosis. We investigated the potential role of a Nox2‐containing NADPH oxidase in aldosterone‐induced fibrosis and the involvement of this mechanism in AngII‐induced effects. Nox2−/− mice were compared with matched wild‐type controls (WT). In WT mice, subcutaneous (s.c.) AngII (1.1 mg/kg/day for 2 wk) significantly increased NADPH oxidase activity, interstitial fibrosis (11.5±1.0% vs. 7.2±0.7%; P<0.05), expression of fibronectin, procollagen I, and connective tissue growth factor mRNA, MMP‐2 activity, and NF‐kB activation. These effects were all inhibited in Nox2−/− hearts. The mineralocorticoid receptor antagonist spironolactone inhibited AngII‐induced increases in NADPH oxidase activity and the increase in interstitial fibrosis. In a model of mineralocorticoid‐dependent hypertension involving chronic aldosterone infusion (0.2 mg/kg/day) and a 1% Na Cl diet (“ALDO”), WT animals exhibited increased NADPH oxidase activity, pro‐fibrotic gene expression, MMP‐2 activity, NF‐kB activation, and significant interstitial cardiac fibrosis (12.0±1.7% with ALDO vs. 6.3±0.3% without; P<0.05). These effects were inhibited in Nox2−/− ALDO mice (e.g., fibrosis 6.8±0.8% with ALDO vs. 5.8±1.0% without ALDO; P=NS). These results suggest that aldosterone‐dependent activation of a Nox2‐containing NADPH oxidase contributes to the profibrotic effect of AngII in the heart as well as the fibrosis seen in mineralocorticoid‐dependent hypertension.—Johar, S., Cave, A. C., Narayanapanicker, A., Grieve, D. J., Shah, A. M. Aldosterone mediates angiotensin II‐induced interstitial cardiac fibrosis via a Nox2‐containing NADPH oxidase. FASEB J. 20, E846–E854 (2006)

Endothelial Nox4 NADPH Oxidase Enhances Vasodilatation and Reduces Blood Pressure In Vivo

Objective—Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H2O2), but its role in vivo remains unclear. Methods and Results—We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H2O2 production and H2O2-induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. Conclusion—Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H2O2 production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.

Involvement of Nox2 NADPH Oxidase in Adverse Cardiac Remodeling After Myocardial Infarction

Oxidative stress plays an important role in the development of cardiac remodeling after myocardial infarction (MI), but the sources of oxidative stress remain unclear. We investigated the role of Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase in the development of cardiac remodeling after MI. Adult Nox2−/− and matched wild-type (WT) mice were subjected to coronary artery ligation and studied 4 weeks later. Infarct size after MI was similar in Nox2−/− and WT mice. Nox2−/− mice exhibited significantly less left ventricular (LV) cavity dilatation and dysfunction after MI than WT mice (eg, echocardiographic LV end-diastolic volume: 75.7±5.8 versus 112.4±12.3 μL; ejection fraction: 41.6±3.7 versus 32.9±3.2%; both P<0.05). Similarly, in vivo LV systolic and diastolic functions were better preserved in Nox2−/− than WT mice (eg, LV dP/dtmax: 7969±385 versus 5746±234 mm Hg/s; LV end-diastolic pressure: 12.2±1.3 versus 18.0±1.8 mm Hg; both P<0.05). Nox2−/− mice exhibited less cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis; reduced increases in expression of connective tissue growth factor and procollagen 1 mRNA; and smaller increases in myocardial matrix metalloproteinase–2 activity than WT mice. These data suggest that the Nox2-containing reduced nicotinamide-adenine dinucleotide phosphate oxidase contributes significantly to the processes underlying adverse cardiac remodeling and contractile dysfunction post-MI.

Ischaemic preconditioning and contractile function: studies with normothermic and hypothermic global ischaemia.

A significant reduction in the extent of cell necrosis or the incidence of reperfusion-induced arrhythmias can be achieved with ischaemic preconditioning. If preconditioning was also found to be effective in protecting against global ischaemia, then this may have significant implications for the preservation of the heart during cardiac surgery. We therefore investigated this phenomenon in relation to recovery of contractile function after global ischaemia in the isolated rat heart. Isolated working rat hearts (n = 6 per group) were perfused aerobically at 37 degrees C for 20 min and contractile function recorded. This was followed by 10 min of aerobic Langendorff perfusion (control hearts) or 5 min global ischaemia (37 degrees C) + 5 min Langendorff reperfusion (preconditioned hearts). The hearts were then subjected to 10, 15, 20 or 25 min of global ischaemia (37 degrees C) and reperfusion (15 min Langendorff + 20 min working) after which function was again assessed. Preconditioning improved functional recovery after all durations of ischaemia. Thus aortic flow after 10, 15, 20 and 25 min of ischaemia and 35 min of reperfusion recovered to 84, 58, 16 and 5%, respectively, in controls and 88, 74, 55 and 20%, respectively, in the preconditioned groups. To assess whether preconditioning was effective in a surgically relevant model of hypothermic ischaemia, the experiments were repeated with longer periods (45, 70, 90, 115, 135 and 160 min) of ischaemia at 20 degrees C. Under these conditions, normothermic preconditioning increase the post-ischaemic recovery of aortic flow after 115, 135 and 160 min of ischaemic (from 36, 20 and 10%, respectively, in controls to 57, 39 and 26%, respectively, in preconditioned hearts). There was no consistent correlation between tissue high energy phosphate content and enhanced post-ischaemic recovery. Thus, we have demonstrated that ischaemic preconditioning can improve contractile function after global ischaemia in the isolated rat heart, we have defined the duration of ischaemia for which it is operative, and we have shown that this protection is additive to that of hypothermia-induced protection during ischaemia. This may have clinical implications for cardiac surgery.

Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart

The experiments described in this paper were designed and performed between 1991 and 1992 when I was in the final year of my Ph.D studentship. I was introduced to preconditioning at my first American Heart Association meeting in 1989 and was fascinated that evolution had devised an endogenous protective mechanism for the heart, which was far more effective than any intervention developed by man. Not surprising really, considering that evolution has been working on stress adaptation for a couple of a million years! Since the aim of my PhD project was to extend the window of preservation for donor hearts prior to transplantation, after the AHA meeting, I began experiments to explore whether ischaemic preconditioning could provide protection over and above that provided by cardioplegia. For the transplanted heart, we felt the most important parameter for assessment was contractile function. For a heart to be weaned off cardiopulmonary bypass, contractile function must recover sufficiently to support the body. It would be somewhat irrelevant for the transplanted heart if preconditioning reduced cell necrosis but left the heart so stunned that it could not maintain a sufficient cardiac output. However, in 1989, there were no published studies specifically investigating preconditioning-induced protection against contractile dysfunction. Therefore, my initial work characterised the phenomenon of preconditioning using the isolated ejecting rat heart and contractile function as the end-point of assessment [1] before proceeding to investigate whether preconditioning could …

Glycated Proteins Stimulate Reactive Oxygen Species Production in Cardiac Myocytes Involvement of Nox2 (gp91phox)-Containing NADPH Oxidase

Background— Nonenzymatic glycation that results in the production of early-glycation Amadori-modified proteins and advanced-glycation end products may be important in the pathogenesis of diabetic complications. However, the effects of early-glycated proteins, such as glycated serum albumin (Gly-BSA), are poorly defined. In this study, we investigated the effects of Gly-BSA on reactive oxygen species (ROS) production by cardiomyocytes. Methods and Results— Cultured neonatal rat cardiomyocytes were incubated with Gly-BSA or vehicle (bovine serum albumin [BSA]) for up to 48 hours. Gly-BSA dose-dependently increased in situ ROS production (whole-cell dichlorodihydrofluorescein fluorescence), with an optimum effect at 400 &mgr;g/mL after 24-hour incubation (152±10% versus BSA 100%; P<0.01). Treatment with the NADPH oxidase inhibitor apocynin, a Nox2 (gp91phox) antisense oligonucleotide (Nox2 AS), or the peptide gp91ds-tat significantly reduced Gly-BSA–induced ROS production at 24 hours (68.5±2.2%, 61.4±8.3%, and 53.2±5.4% reduction, respectively). NADPH-dependent activity in cell homogenates was also significantly increased by Gly-BSA at 24 hours (161±8% versus BSA) and was inhibited by diphenyleneiodonium, apocynin, NOX2AS, and the protein kinase C inhibitor bisindolylmaleimide I but not by a nitric oxide synthase inhibitor or mitochondrial inhibitors. Furthermore, bisindolylmaleimide I prevented Gly-BSA–stimulated Rac1 translocation, an essential step for NADPH oxidase activation. Gly-BSA–induced increases in ROS were associated with apocynin-inhibitable nuclear translocation of nuclear factor-&kgr;B and an increase in atrial natriuretic factor mRNA expression. Conclusions— Gly-BSA stimulates cardiomyocyte ROS production through a protein kinase C–dependent activation of a Nox2-containing NADPH oxidase, which results in nuclear factor-&kgr;B activation and upregulation of atrial natriuretic factor mRNA. These findings suggest that early-glycated Amadori products may play a role in the development of diabetic heart disease.

Pivotal role of NOX-2-containing NADPH oxidase in early ischemic preconditioning

Reactive oxygen species (ROS)‐mediated signaling is implicated in early ischemic preconditioning (PC). A NOX‐2‐containing NADPH oxidase is a recognized major source of ROS in cardiac myocytes, whose activity is augmented by preconditioning mimetics, such as angiotensin II. We hypothesized that this oxidase is an essential source of ROS in PC. Hearts from wild‐type (WT) and NOX‐2 knockout (KO) mice were Langendorff perfused and subjected to 35 min ischemia/reperfusion with or without preceding PC or drug treatment. Infarct size was measured by triphenyl tetrazolium chloride staining, and NADPH oxidase activity by lucigenin chemiluminescence. PC significantly attenuated infarct size in WT (26±2% vs. control, 38±2%, P<0.05) yet was ineffective in KO hearts (33±3% vs. control, 34±3%). Concomitantly, PC significantly increased NADPH oxidase activity in WT (+41±13%; P<0.05), but not in KO (−5±18%, P=NS). The ROS scavenger MPG (N‐2‐mercaptopropionyl glycine, 300 µmol/L) abrogated PC in WT (39±2% vs. control, 33±1%). CCPA (2‐chloro N6 cyclopentyl adenosine, 200 nmol/L), a putative ROS‐independent PC trigger, significantly attenuated infarct size in WT, MPG‐treated WT and KO hearts (24±2, 23±1, and 20±3%, respectively, P<0.05). Furthermore, CCPA did not augment NADPH oxidase activity over control (+22±11%, P=NS). Inhibition of protein kinase C (PKC) with chelerythrine (CHE, 2 µmol/L) completely abrogated both PC (38±2% vs. CHE alone, 35±2%) and associated increases in oxidase activity (+3±10%, P=NS). PKC‐dependent activation of a NOX‐2‐containing NADPH oxidase is pivotally involved in early ischemic PC. However, adenosine receptor activation can trigger a ROS and NOX‐2 independent PC pathway.

PET and NMR dual acquisition (PANDA): Applications to isolated, perfused rat hearts

Positron emission tomography and nuclear magnetic resonance spectroscopy are non‐invasive techniques that allow serial metabolic measurements to be obtained in a single subject. Significant advantages could be obtained if both types of scans could be acquired with a single machine. A small‐scale PET scanner, designed to operate in a high magnetic field, was therefore constructed and inserted into the top half of a 7.3 cm bore, 9.4 T NMR magnet and its performance characterized. The magnetic field did not significantly affect either the sensitivity (∼ 3 kcps/MBq) or the spatial resolution (2.0 mm full width at half maximum, measured using a 0.25 mm diameter line source) of the scanner. However, the presence of the PET scanner resulted in a small decrease in field homogeneity. The first, simultaneous 31P NMR spectra (200, 80° pulses collected at 6 s intervals) and PET images (transverse, mid‐ventricular slices at the level of the mitral value) from isolated, perfused rat hearts were acquired using a specially designed NMR probe inserted into the bottom half of the magnet. The PET images were of excellent quality, enabling the left ventricular wall and interventricular septum to be clearly seen. In conclusion, we have demonstrated the simultaneous acquisition of PET and NMR data from perfused rat hearts; we believe that the combination of these two powerful techniques has tremendous potential in both the laboratory and the clinic.