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

Department of Physiology, New York Medical College, Valhalla, New York 10595 Submitted 3 December 2002 ; accepted in final form 8 May 2003 We investigated the role of nitric oxide (NO) in the control of myocardial O 2 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 O 2 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%at4moto66 ± 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 O 2 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 O 2 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 p67 phox , however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91 phox , and apocynin restored the defect in NO-dependent control of cardiac O 2 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 O 2 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. nitric oxide; Western blotting; tempol; mitochondria; SOD1; SOD2; SOD3; p67 phox ; gp91 phox ; apocynin Address for reprint requests and other correspondence: T. H. Hintze, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail: thomas_h