Barbara Casadei

A Myocardial Nox2 Containing NAD(P)H Oxidase Contributes to Oxidative Stress in Human Atrial Fibrillation

Human atrial fibrillation (AF) has been associated with increased atrial oxidative stress. In animal models, inhibition of reactive oxygen species prevents atrial remodeling induced by rapid pacing, suggesting that oxidative stress may play an important role in the pathophysiology of AF. NAD(P)H oxidase is a major source of superoxide in the cardiovascular system; however, whether this enzyme contributes to atrial oxidative stress in AF remains to be elucidated. We investigated the sources of superoxide production (using inhibitors and substrates of a range of oxidases, RT-PCR, immunofluorescence, and immunoblotting) in tissue homogenates and isolated atrial myocytes from the right atrial appendage (RAA) of patients undergoing cardiac surgery (n=54 in sinus rhythm [SR] and 15 in AF). A membrane-bound gp91phox containing NAD(P)H oxidase in atrial myocytes was the main source of atrial superoxide production in SR and in AF. NADPH-stimulated superoxide release from RAA homogenates was significantly increased in patients with AF in the absence of changes in mRNA expression of the p22phox and gp91phox subunits of the NAD(P)H oxidase. In contrast with findings in SR patients, NO synthases (NOSs) contributed significantly to atrial superoxide production in fibrillating atria, suggesting that increased oxidative stress in AF may lead to NOS “uncoupling.” These findings indicate that a myocardial NAD(P)H oxidase and, to a lesser extent, dysfunctional NOS contribute significantly to superoxide production in the fibrillating human atrial myocardium and may play an important role in the atrial oxidative injury and electrophysiological remodeling observed in patients with AF.

Nitric Oxide Can Increase Heart Rate by Stimulating the Hyperpolarization-Activated Inward Current, If

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n = 8) or 3-morpholinosydnonimine (SIN-1, n = 6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n = 6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 mumol/L, n = 6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 mumol/L, n = 6), and (3) mimicked by 8-bromo-cGMP (n = 6) with no additional positive chronotropic effect of SIN-1 (n = 5). The response to 10 mumol/L SNP (n = 28) or 50 mumol/L SIN-1 (n = 16) was unaffected by IcaL antagonism with nifedipine (0.2 mumol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I(f)) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 mumol/L). The effect on I(f) was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n = 21), where we found that 5 mumol/L SNP or SIN-1 caused a reversible Cs(+)-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I(f) via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.

Determinants of Spontaneous Baroreflex Sensitivity in a Healthy Working Population

Baroreflex sensitivity (BRS) by the spontaneous sequence technique has been widely used as a cardiac autonomic index for a variety of pathological conditions. However, little information is available on determinants of the variability of spontaneous BRS and on age-related reference values of this measurement in a healthy population. We evaluated BRS as the slope of spontaneous changes in systolic blood pressure (BP) and pulse interval from 10 minutes BP (Finapres) and ECG recordings in 1134 healthy volunteers 18 to 60 years of age. Measurement of BRS could be obtained in 90% of subjects. Those with unmeasurable spontaneous BRS had a slightly lower heart rate but were otherwise not different from the rest of the population. BRS was inversely related to age (lnBRS, 3.24−0.03×age;r2=0.23;P <0.0001) in both genders. In addition, univariate analysis revealed a significant inverse correlation between BRS and heart rate, body mass index, and BP. Sedentary lifestyle and regular alcohol consumption were also associated with lower BRS. However, only age, heart rate, systolic and diastolic BP, body mass index, smoking, and gender were independent predictors of BRS in a multivariate model, accounting for 47% of the variance of BRS. The present study provides reference values for spontaneous BRS in a healthy white population. Only approximately half of the variability of BRS could be explained by anthropometric variables and common risk factors, which suggests that a significant proportion of interindividual differences may reflect genetic heterogeneity.

Cardiac Nitric Oxide Synthase 1 Regulates Basal and β-Adrenergic Contractility in Murine Ventricular Myocytes

Background—Evidence indicates that myocardial NO production can modulate contractility, but the source of NO remains uncertain. Here, we investigated the role of a type 1 NO synthase isoform (NOS1), which has been recently localized to the cardiac sarcoplasmic reticulum, in the regulation of basal and &bgr;-adrenergic myocardial contraction. Methods and Results—Contraction was assessed in left ventricular myocytes isolated from mice with NOS1 gene disruption (NOS1−/− mice) and their littermate controls (NOS1+/+ mice) at 3 stimulation frequencies (1, 3, and 6 Hz) in basal conditions and during &bgr;-adrenergic stimulation with isoproterenol (2 nmol/L). In addition, we examined the effects of acute specific inhibition of NOS1 with vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (L-VNIO, 500 &mgr;mol/L). NOS1−/− myocytes exhibited greater contraction at all frequencies (percent cell shortening at 6 Hz, 10.7±0.92% in NOS1−/− myocytes versus 7.21±0.8% in NOS1+/+ myocytes;P <0.05) with a flat frequency-contraction relationship. Time to 50% relaxation was increased in NOS1−/− myocytes at all frequencies (at 6 Hz, 26.53±1.4 ms in NOS1−/− myocytes versus 21.27±1.3 ms in NOS1+/+ myocytes;P <0.05). L-VNIO prolonged time to 50% relaxation at all frequencies (at 6 Hz, 21.28±1.7 ms in NOS1+/+ myocytes versus 26.45±1.4 ms in NOS1+/++L-VNIO myocytes;P <0.05) but did not significantly increase basal contraction. However, both NOS1−/− myocytes and NOS1+/+ myocytes treated with L-VNIO showed a greatly enhanced contraction in response to &bgr;-adrenergic stimulation (percent increase in contraction at 6 Hz, 25.2±10.8 in NOS1+/+ myocytes, 68.2±11.2 in NOS1−/− myocytes, and 65.1±13.2 in NOS1+/++L-VNIO myocytes;P <0.05). Conclusions—NOS1 disruption enhances basal contraction and the inotropic response to &bgr;-adrenergic stimulation in murine ventricular myocytes. These findings indicate that cardiac NOS1-derived NO plays a significant role in the autocrine regulation of myocardial contractility.

Rapid, Direct Effects of Statin Treatment on Arterial Redox State and Nitric Oxide Bioavailability in Human Atherosclerosis via Tetrahydrobiopterin-Mediated Endothelial Nitric Oxide Synthase Coupling

Background— Treatment with statins improves clinical outcome, but the exact mechanisms of pleiotropic statin effects on vascular function in human atherosclerosis remain unclear. We examined the direct effects of atorvastatin on tetrahydrobiopterin-mediated endothelial nitric oxide (NO) synthase coupling in patients with coronary artery disease. Methods and Results— We first examined the association of statin treatment with vascular NO bioavailability and arterial superoxide (O2·−) in 492 patients undergoing coronary artery bypass graft surgery. Then, 42 statin-naïve patients undergoing elective coronary artery bypass graft surgery were randomized to atorvastatin 40 mg/d or placebo for 3 days before surgery to examine the impact of atorvastatin on endothelial function and O2·− generation in internal mammary arteries. Finally, segments of internal mammary arteries from 26 patients were used in ex vivo experiments to evaluate the statin-dependent mechanisms regulating the vascular redox state. Statin treatment was associated with improved vascular NO bioavailability and reduced O2·− generation in internal mammary arteries. Oral atorvastatin increased vascular tetrahydrobiopterin bioavailability and reduced basal and N-nitro-L-arginine methyl ester–inhibitable O2·− in internal mammary arteries independently of low-density lipoprotein lowering. In ex vivo experiments, atorvastatin rapidly improved vascular tetrahydrobiopterin bioavailability by upregulating GTP-cyclohydrolase I gene expression and activity, resulting in improved endothelial NO synthase coupling and reduced vascular O2·−. These effects were reversed by mevalonate, indicating a direct effect of vascular hydroxymethylglutaryl-coenzyme A reductase inhibition. Conclusions— This study demonstrates for the first time in humans the direct effects of statin treatment on the vascular wall, supporting the notion that this effect is independent of low-density lipoprotein lowering. Atorvastatin directly improves vascular NO bioavailability and reduces vascular O2·− through tetrahydrobiopterin-mediated endothelial NO synthase coupling. These findings provide new insights into the mechanisms mediating the beneficial vascular effects of statins in humans. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01013103.

Identification of higher brain centres that may encode the cardiorespiratory response to exercise in humans

1 Positron emission tomography (PET) was used to identify the neuroanatomical correlates underlying ‘central command’ during imagination of exercise under hypnosis, in order to uncouple central command from peripheral feedback. 2 Three cognitive conditions were used: condition I, imagination of freewheeling downhill on a bicycle (no change in heart rate, HR, or ventilation, V̇I): condition II, imagination of exercise, cycling uphill (increased HR by 12 % and V̇I by 30 % of the actual exercise response): condition III, volitionally driven hyperventilation to match that achieved in condition II (no change in HR). 3 Subtraction methodology created contrast A (II minus I) highlighting cerebral areas involved in the imagination of exercise and contrast B (III minus I) highlighting areas activated in the direct volitional control of breathing (n= 4 for both; 8 scans per subject). End‐tidal PCO2 (PET,CO2) was held constant throughout PET scanning. 4 In contrast A, significant activations were seen in the right dorso‐lateral prefrontal cortex, supplementary motor areas (SMA), the right premotor area (PMA), superolateral sensorimotor areas, thalamus, and bilaterally in the cerebellum. In contrast B, significant activations were present in the SMA and in lateral sensorimotor cortical areas. The SMA/PMA, dorso‐lateral prefrontal cortex and the cerebellum are concerned with volitional/motor control, including that of the respiratory muscles. 5 The neuroanatomical areas activated suggest that a significant component of the respiratory response to ‘exercise’, in the absence of both movement feedback and an increase in CO2 production, can be generated by what appears to be a behavioural response.

Association of Atrial Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity With the Development of Atrial Fibrillation After Cardiac Surgery

OBJECTIVES Our goal was to evaluate the role of myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and plasma markers of oxidative stress in the pathogenesis of post-operative atrial fibrillation (AF). BACKGROUND Atrial fibrillation is a common complication of cardiac surgery, leading to increased morbidity and prolonged hospitalization. Experimental evidence suggests that oxidative stress may be involved in the pathogenesis of AF; however, the relevance of this putative mechanism in patients undergoing cardiac surgery is unclear. METHODS We measured basal and NADPH-stimulated superoxide production in right atrial appendage samples from 170 consecutive patients undergoing conventional coronary artery bypass surgery. Plasma markers of lipid and protein oxidation (thiorbabituric acid-reactive substances, 8-isoprostane, and protein carbonyls) were also measured in blood samples drawn from a central line before surgery and after reperfusion. RESULTS Patients who developed AF after surgery (42%) were older and had a significantly increased atrial NADPH oxidase activity than patients who remained in sinus rhythm (SR) (in relative light units/s/mug protein: 4.78 +/- 1.44 vs. 3.53 +/- 1.04 in SR patients, p < 0.0001). Plasma markers of lipid and protein oxidation increased significantly after reperfusion; however, neither pre-operative nor post-operative measurements differed between patients who developed AF and those who remained in SR after surgery. Multivariate analysis identified atrial NADPH oxidase activity as the strongest independent predictor of post-operative AF (odds ratio 2.41; 95% confidence interval 1.71 to 3.40, p < 0.0001). CONCLUSIONS Atrial NADPH oxidase activity is independently associated with an increased risk of post-operative AF, suggesting that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after cardiac surgery.

Interactions Between Vascular Wall and Perivascular Adipose Tissue Reveal Novel Roles for Adiponectin in the Regulation of Endothelial Nitric Oxide Synthase Function in Human Vessels

Background— Adiponectin is an adipokine with potentially important roles in human cardiovascular disease states. We studied the role of adiponectin in the cross-talk between adipose tissue and vascular redox state in patients with atherosclerosis. Methods and Results— The study included 677 patients undergoing coronary artery bypass graft surgery. Endothelial function was evaluated by flow-mediated dilation of the brachial artery in vivo and by vasomotor studies in saphenous vein segments ex vivo. Vascular superoxide (O2−) and endothelial nitric oxide synthase (eNOS) uncoupling were quantified in saphenous vein and internal mammary artery segments. Local adiponectin gene expression and ex vivo release were quantified in perivascular (saphenous vein and internal mammary artery) subcutaneous and mesothoracic adipose tissue from 248 patients. Circulating adiponectin was independently associated with nitric oxide bioavailability and O2− production/eNOS uncoupling in both arteries and veins. These findings were supported by a similar association between functional polymorphisms in the adiponectin gene and vascular redox state. In contrast, local adiponectin gene expression/release in perivascular adipose tissue was positively correlated with O2− and eNOS uncoupling in the underlying vessels. In ex vivo experiments with human saphenous veins and internal mammary arteries, adiponectin induced Akt-mediated eNOS phosphorylation and increased tetrahydrobiopterin bioavailability, improving eNOS coupling. In ex vivo experiments with human saphenous veins/internal mammary arteries and adipose tissue, we demonstrated that peroxidation products produced in the vascular wall (ie, 4-hydroxynonenal) upregulate adiponectin gene expression in perivascular adipose tissue via a peroxisome proliferator-activated receptor-&ggr;–dependent mechanism. Conclusions— We demonstrate for the first time that adiponectin improves the redox state in human vessels by restoring eNOS coupling, and we identify a novel role of vascular oxidative stress in the regulation of adiponectin expression in human perivascular adipose tissue.

Prediction of patient responses to antihypertensive drugs using genetic polymorphisms : investigation of renin-angiotensin system genes

OBJECTIVE To investigate whether the M235T polymorphism of the angiotensinogen (AGT) gene and the insertion/deletion (I/D) polymorphism of the angiotensin-1 converting enzyme (ACE) gene predict blood pressure response to different antihypertensive agents. DESIGN Sixty-three patients with untreated essential hypertension were randomly assigned in a placebo-controlled crossover comparison to atenolol 50 mg once daily, lisinopril 10 mg once daily and nifedipine SR 20 mg twice daily, and the effect on blood pressure was assessed by ambulatory blood pressure monitoring (ABPM). In a further 44 patients, placebo-controlled ABPM data were available after treatment with a single agent (atenolol 50 mg once daily in 16 cases and lisinopril 10mg once daily in 28 cases). The change in systolic and diastolic blood pressure achieved by each agent was analysed for association with genotypes at the AGT and ACE gene loci. METHODS Polymerase chain reaction (PCR) amplification of genomic DNA from each individual was used to identify the I/D polymorphism of the ACE gene. The M235T polymorphism of the AGT gene was detected by Tth111I digestion of PCR product. RESULTS There was no significant association between response to any drug and either the AGT M235T or ACE I/D polymorphisms. CONCLUSIONS The large variability between individuals in the observed blood pressure response to these agents cannot be attributed to the polymorphisms analysed at the ACE and AGT loci.

Preoperative Atorvastatin Treatment in CABG Patients Rapidly Improves Vein Graft Redox State by Inhibition of Rac1 and NADPH-Oxidase Activity

Background— Statins improve clinical outcome of patients with atherosclerosis, but their perioperative role in patients undergoing coronary artery bypass grafting (CABG) is unclear. We hypothesized that short-term treatment with atorvastatin before CABG would improve the redox state in saphenous vein grafts (SVGs), independently of low-density lipoprotein cholesterol (LDL)-lowering. Methods and Results— In a randomized, double-blind controlled trial, 42 statin-naïve patients undergoing elective CABG received atorvastatin 40 mg/d or placebo for 3 days before surgery. Circulating inflammatory markers and malondialdehyde (MDA) were measured before and after treatment. SVG segments were used to determine vascular superoxide (O2·−) and Rac1 activation. For ex vivo studies, SVG segments from 24 patients were incubated for 6 hours with atorvastatin 0, 5, or 50 &mgr;mol/L. Oral atorvastatin reduced vascular basal and NADPH-stimulated O2·− in SVGs (P<0.05 for all versus placebo) and reduced plasma MDA (P<0.05), independently of LDL-lowering and of changes in inflammatory markers. In SVGs exposed to atorvastatin ex vivo, without exposure to LDL, basal and NADPH-stimulated O2·− were significantly reduced (P<0.01 for both concentrations versus 0 &mgr;mol/L) in association with a striking reduction in Rac1 activation and 1 membrane-bound Rac1 and p67phox subunit. The antioxidant effects of atorvastatin were reversed by mevalonate, implying a dependence on vascular HMG-CoA reductase inhibition. Conclusions— Short-term treatment with atorvastatin 40 mg/d before CABG improves redox state in SVGs, by inhibiting vascular Rac1-mediated activation of NADPH-oxidase. These novel findings suggest that statin therapy should be maintained or initiated in patients undergoing CABG, independently of LDL levels. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01013103.