NAD(P)H oxidase-generated superoxide anion accounts for reduced control of myocardial O₂ consumption by NO in old Fischer 344 rats

Abstract

We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer 344 rats. In Fischer rats at 4, 14, and 23 mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 +/- 2% at 4 mo to 66 +/- 3% (P < 0.05) at 23 mo] and an increased cardiac diastolic volume [from 0.60 +/- 0.04 to 1.00 +/- 0.10 ml (P < 0.01)] and heart weight (from 0.70 +/- 0.02 to 0.90 +/- 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (36 +/- 2 or 34 +/- 3%) and 14 mo (29 +/- 1 or 25 +/- 3%) but markedly (P < 0.05) reduced in 23-mo-old Fischer rats (15 +/- 3 or 7 +/- 2%). The response to the NO donor S-nitroso-N-acetyl penicillamine was not different across groups (35%, 35%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 23 mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 +/- 3%) or enalaprilat (28 +/- 3%) in 23-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn SOD, Cu/Zn SOD, extracellular SOD, and p67phox, however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 23 mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 23-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.

Limited Exercise Capacity in Heterozygous Manganese Superoxide Dismutase Gene–Knockout Mice

“…26 Furthermore, we reported that NO-dependent control of V O 2 was attenuated in cardiac muscle from SOD2 ϩ/Ϫ mice, which was reversed by the freely membrane-permeable O 2 Ϫ scavenger Tiron. 17 Our previous in vitro studies 17,25,26 have shown that O 2 Ϫ plays an important role in the regulation of NOdependent control of V O 2 . These studies support our present finding that increased O 2 Ϫ in skeletal muscle reduces NO bioavailability and NO-dependent control of V O 2 in vivo.…”

“…NO-dependent control of V O 2 in cardiac muscle from old Fisher 344 rats, a model of accelerated aging, was reduced and was associated with NAD(P)H oxidase-generated O 2 Ϫ . 25 Angiotensin II at pathophysiological concentrations stimulates an increase in O 2 Ϫ through the activation of NAD(P)H oxidase and attenuates NO-dependent control of V O 2 in cardiac muscle from normal dogs. 26 Furthermore, we reported that NO-dependent control of V O 2 was attenuated in cardiac muscle from SOD2 ϩ/Ϫ mice, which was reversed by the freely membrane-permeable O 2 Ϫ scavenger Tiron.…”

“…V O 2 in skeletal muscle in vitro was measured as we described previously. 17,[25][26][27] The muscle tissues (about 50 mg) were incubated in Krebs solution (mol/L: 118 NaCl, 4.7 KCl, 1.5 CaCl 2 , 25 NaHCO 3 , 1.1 MgSO 4 , 1.2 KH 2 PO4, and 5.6 glucose) at 37°C for 2 hours and bubbled continuously with 20% O 2 -5% CO 2 -75% N 2 . At the end of the incubation period, each piece of tissue was placed in a stirred bath with 3 mL of air-saturated Krebs bicarbonate solution containing 10 mmol/L HEPES (pH 7.4).…”

Vascular Dysfunction in Aging: Potential Effects of Resveratrol, an Anti- Inflammatory Phytoestrogen

“…Additional studies have also described increased ROS production in the carotid arteries and aorta of aged rats [41,42] and mice [43]. Aging-induced vascular oxidative stress is characterized by a down-regulation of antioxidants, such as ecSOD [40], an increased expression of iNOS [1,43] and increased activity of NAD(P)H oxidases [7,42,44] and/or other oxidase mechanisms [45]. Increased oxidative stress in aging leads to functional inactivation of NO via increased O 2 ·− levels yielding in an increase in formation of a reactive oxidant ONOO − , which may impair cardiovascular function through multiple mechanisms [7,40,43,44,46,47].…”

Aging might augment reactive oxygen species (ROS) formation and affect reactive nitrogen species (RNS) level after myocardial ischemia/reperfusion in both humans and rats

“…·O 2 − further dismutates to H 2 O 2 and ·OH, the latter being highly toxic to biological tissues, causative of significant myocardial necrosis and apoptosis. Several recent studies report that aging increases reactive oxygen species (ROS) (Adler et al 2003;Jacob et al 2010a;Jacob et al 2010b;Judge et al 2005). Leeuwenburgh and colleagues demonstrate increased mitochondrial ROS formation with aging (Judge et al 2005).…”