Abstract-Inhibition of xanthine oxidase (XO) in failing hearts improves cardiac efficiency by an unknown mechanism.We hypothesized that this energetic effect is due to reduced oxidative stress and critically depends on nitric oxide synthase (NOS) activity, reflecting a balance between generation of nitric oxide (NO) and reactive oxygen species. In dogs with pacing-induced heart failure (HF), ascorbate (1000 mg) mimicked the beneficial energetic effects of allopurinol, increasing both contractility and efficiency, suggesting an antioxidant mechanism. Allopurinol had no additive effect beyond that of ascorbate. Crosstalk between XO and NOS signaling was assessed. NOS inhibition with N G -monomethyl-L-arginine (L-NMMA; 20 mg/kg) had no effect on basal contractility or efficiency in HF, but prevented the ϩ26.2Ϯ3.5% and ϩ66.5Ϯ17% enhancements of contractility and efficiency, respectively, observed with allopurinol alone. Similarly, improvements in contractility and energetics due to ascorbate were also inhibited by L-NMMA. Because of the observed NOS-XO crosstalk, we predicted that in normal hearts NOS inhibition would uncover a depression of energetics caused by XO activity. In normal conscious dogs, L-NMMA increased myocardial oxygen consumption (MV O 2 ) while lowering left ventricular external work, reducing efficiency by 31.1Ϯ3.8% (PϽ0.005). Lowered efficiency was reversed by XO inhibition (allopurinol, 200 mg) or by ascorbate without affecting cardiac load or systemic hemodynamics. Single-cell immunofluorescence detected XO protein in cardiac myocytes that was enhanced in HF, consistent with autocrine signaling. These data show that both NOS and XO signaling systems participate in the regulation of myocardial mechanical efficiency and that upregulation of XO relative to NOS contributes to mechanoenergetic uncoupling in heart failure. Key Words: xanthine oxidase Ⅲ oxidative stress Ⅲ nitric oxide Ⅲ heart failure Ⅲ ascorbate T he failing heart displays substantial energetic inefficiency in both isolated muscle 1,2 and intact chambers. [3][4][5] This phenomenon can be best described as "mechanoenergetic uncoupling," given that the depression of contractile force is not matched by a concomitant depression of energy consumption. Among the proposed mechanisms is enhanced oxidative stress stemming from mitochondrial 6 and cytosolic free radical generating systems. 7 Xanthine oxidase (XO) is prominent among these enzymes, because it produces superoxide as a byproduct of the terminal two steps of purine metabolism. 3 XO is upregulated in failing myocardium of experimental animals 3,8 and humans, 9 and its inhibition by allopurinol improves the mechanical efficiency (the ratio between ventricular work performed and oxygen consumed) of intact failing hearts 3,9 ; such an effect was predicted by the initial observations that allopurinol and oxypurinol augment calcium-activated force without increasing activator Ca 2ϩ in isolated cardiac muscle. 2 To date, however, the signal transduction mechanisms of the salutary effects o...

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It is generally accepted that inhibition of nitric oxide synthase (NOS) facilitates, and thus nitric oxide (NO) inhibits, contractility of skeletal muscle. However, standard assessments of contractility are carried out at a nonphysiological oxygen tension [partial pressure of oxygen (pO2)] that can interfere with NO signaling (95% O2). We therefore examined, in normal and neuronal NOS (nNOS)-deficient mice, the influence of pO2 on whole-muscle contractility and on myocyte calcium flux and sarcomere shortening. Here, we demonstrate a significant enhancement of these measures of muscle performance at low physiological pO2 and an inhibitory influence at higher physiological pO2, which depend on endogenous nNOS. At 95% O2 (which produces oxidative stress; muscle core pO2 Ϸ400 mmHg), force production is enhanced but control of contractility by NO͞nitrosylation is greatly attenuated. In addition, responsivity to pO2 is altered significantly in nNOS mutant muscle. These results reveal a fundamental role for the concerted action of NO and O2 in physiological regulation of skeletal muscle contractility, and suggest novel molecular aspects of myopathic disease. They suggest further that the role of NO in some cellular systems may require reexamination. It has recently been recognized that redox-based regulation of protein function serves not only to mediate compensatory responses to oxidative or nitrosative stress but also modulates transduction along basic signaling pathways in mammalian cells (1). Further, accumulating evidence indicates that nitric oxide (NO)-based protein modifications are critical effectors of redox regulation, which may be linked at the molecular level to partial pressure of oxygen (pO 2 ) (1). Accordingly, the functional contribution of redox mechanisms remains, for the most part, untested, and potentially masked in virtually all ex vivo studies, which have been carried out at supranormal oxygen tension (typically 21% O 2 for cells and 95% O 2 for tissue, whereas pO 2 in vivo is much lower).Mammalian skeletal muscle operates over a range of pO 2 , which is determined by local blood flow and muscle activity, and contains endogenous sources of NO. The -isoform of type I or neuronal NO synthase (nNOS) localizes to the plasma membrane (sarcolemma) of skeletal muscle fibers through interaction with the dystrophin complex (2-4). In many mammals, nNOS is either restricted to or particularly abundant in fast-twitch fibers, although, in humans, fast-and slow-twitch fibers are more evenly endowed (2, 5, 6). The loss of nNOS from sarcolemma of mdx mice (whose fast-twitch fibers are disproportionately impaired), and of patients with Duchenne or Becker muscular dystrophy, focused interest on the possibility that NO produced by muscle fibers may play a role in excitation-contraction (E-C) coupling and that NO deficiency may contribute to contractile impairment in muscle disease (2-6). However, although mice deficient in nNOS show subtle abnormalities in muscle blood flow (7), overt alterations in contractile fun...