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Tonic activation of adrenergic drive has been found to be associated with aging, and its further activation is also seen in aging patients with major surgery or congestive heart failure. Nevertheless, its potential effect on the aging heart remains enigmatic. In the present study, at baseline, significant inflammatory and apoptotic changes were found in the aging mouse (20 months old), as evidenced by increases in inducible nitric oxide synthase (iNOS) expression, myocardial apoptosis in the heart, and C-reactive protein (CRP) release in the circulation. These phenotypic changes in aging animals can be induced in young animals (3 months old) by chronic beta-adrenergic receptor (AR) stimulation with isoproterenol (ISO), and they can be markedly reduced in aging animals by chronic beta-blockade with propranolol. Compared with young animals, chronic beta-AR stimulation with ISO in aging animals induced larger increases in iNOS expression, nitrotyrosine formation in the heart, and nitric oxide (NO) production and CRP release in the circulation; it also accelerated myocardial apoptosis and resulted in an enlarged infarct size when animals were subjected to myocardial ischemia and reperfusion (MI/R). However, the pretreatment of 1400W (N-(3-(aminomethyl) benzyl)acetamidine)-a specific iNOS inhibitor-significantly reduced iNOS-mediated nitrative stress associated with a marked decrease in myocardial apoptosis and infarct size in aging mice. These results demonstrate that tonic activation of the beta-adrenergic system associated with aging induces proinflammatory and proapoptotic changes in the heart and that additional beta-AR stimulation results in an exaggerated nitrative stress, mediated by iNOS, that is associated with more severe myocardial injury in aging mice.

The present study provides evidence that inducible nitric-oxide synthase (iNOS)-mediated nitrative stress plays a pivotal role in chronic beta-adrenergic receptor (AR) stimulation-induced cardiac damage. In mice, 14 days of isoproterenol (ISO) stimulation via an osmotic minipump induced an up-regulation of iNOS as evidenced by increases in mRNA, protein expression, and immunochemical staining of myocardial iNOS. Serum level of C-reactive protein, an inflammatory mediator, was also markedly increased. Under chronic ISO stimulation, the up-regulated iNOS produced a significantly increased amount of nitric oxide (NO) and its byproduct, peroxynitrite, in the circulation and heart and subsequently resulted in an accelerated myocardial apoptosis. Forty-minute myocardial ischemia (MI) and 24-h reperfusion (R) further increased NO production and peroxynitrite formation and resulted in an enlarged infarct size in mice receiving chronic ISO stimulation. However, the treatment with a selective iNOS inhibitor [N-(3-(aminomethyl) benzyl)acetamidine] (1400W) or the use of a genetic modified animal (iNOS-knockout mice) markedly reduced iNOS-mediated production of NO and formation of peroxynitrite and consequently significantly decreased myocardial apoptosis and infarct size, showing a crucial link between iNOS-mediated nitrative stress and myocardial injury. In conclusion, chronic beta-AR stimulation up-regulates iNOS expression and increases NO production in the heart, which subsequently markedly enhances formation of reactive nitrogen species/peroxynitrite in the heart, thereby eliciting myocardial apoptosis and potentiating MI/R injury.

We have previously shown in dogs that the hydrophilic alpha-tocopherol analog, MDL 74,405, attenuates postischemic myocardial dysfunction ("stunning") and generation of free radicals as assessed with the spin trap alpha-phenyl N-tert-butyl nitrone (PBN). However, we could not discern whether this drug acts on primary radicals (such as hydroxyl radical [.OH]) or on secondary radicals. The goal of this study was to directly determine whether the beneficial effects of MDL 74,405 result from actions against .OH. Open-chest dogs undergoing a 15-minute coronary artery occlusion and 3 hours of reperfusion received an intravenous infusion of either saline solution (control group, n = 7) or MDL 74,405 (n = 6) starting 30 minutes before coronary occlusion and ending 60 minutes after reflow at a dose of 0.3 mg/kg/hr. Formation of .OH was estimated by the technique of aromatic hydroxylation of phenylalanine. Phenylalanine was infused intravenously, and the plasma concentrations of the hydroxylated products ortho-, meta-, and para-tyrosines (o-, m-, and p-tyr) in the coronary venous effluent and in the arterial blood were measured with high-performance liquid chromatography. In the control group a dramatic increase in the myocardial release of o-, m-, and p-tyr was observed immediately after reperfusion; the release of tyrosines peaked at 1 minute of reflow and continued up to 10 minutes after reperfusion. MDL 74,405 abolished the release of o-tyr throughout the first 10 minutes of reperfusion but had a less pronounced effect on the production of m- and p-tyr. These results demonstrate that MDL 74,405 is effective in inhibiting .OH-initiated reactions in the postischemic stunned myocardium in the dog, suggesting that the anti-.OH action of MDL 74,405 is an important mechanism of action of this antioxidant.

Conscious pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, for three consecutive days (days 1, 2, and 3 of stage I). The recovery of systolic wall thickening (WTh) after the 10th reperfusion was markedly improved on days 2 and 3 compared with day 1, indicating that the myocardium had become preconditioned against "stunning." 10 d after stage I, pigs underwent again a sequence of 10 2-min coronary occlusions for two consecutive days (days 1 and 2 of stage II). On day 1 of stage II, the recovery of WTh after the 10th reperfusion was similar to that noted on day 1 of stage I; on day 2 of stage II, however, the recovery of WTh was again markedly improved compared with day 1. Blockade of adenosine receptors with 8-p-sulfophenyl theophylline failed to prevent the development of preconditioning against stunning. Northern blot analysis demonstrated an increase in heat stress protein (HSP) 70 mRNA 2 h after the preconditioning ischemia; at this same time point, immunohistochemical analysis revealed a concentration of HSP70 in the nucleus and an overall increase in staining for HSP70. 24 h after the preconditioning ischemia, Western dot blot analysis demonstrated an increase in HSP70. This study indicates the existence of a new, previously unrecognized cardioprotective phenomenon. The results demonstrate that a brief ischemic stress induces a powerful, long-lasting (at least 48 h) adaptive response that renders the myocardium relatively resistant to stunning 24 h later (late preconditioning against stunning). This adaptive response disappears within 10 d after the last ischemic stress but can be reinduced by another ischemic stress. Unlike early and late preconditioning against infarction, late preconditioning against stunning is not blocked by adenosine receptor antagonists, and therefore appears to involve a mechanism different from that of other forms of preconditioning currently known. The increase in myocardial HSP70 is compatible with, but does not prove, a role of HSPs in the pathogenesis of this phenomenon.

Alpha-phenyl N-tert-butyl nitrone (PBN) is widely used in spin-trapping experiments, but its possible toxicity has not been systematically evaluated. The purpose of this study was to investigate the effects of different doses of PBN on cardiac function in vivo (open-chest dogs) and in vitro (isolated rat hearts). In open-chest dogs, PBN was infused intracoronarily to achieve coronary arterial concentrations ranging from 1.6 mM to 10.0 mM. At coronary arterial concentrations of 1.6 mM and 2.5 mM, PBN had no appreciable effect on regional myocardial function (assessed as systolic wall thickening). However, coronary arterial concentrations of PBN of 5.0 mM and 10.0 mM produced a marked reduction and, eventually, a complete loss of systolic wall thickening (53% of baseline values after 30 min at 5.0 mM and 14% after 30 min at 10.0 mM). Furthermore, PBN increased coronary blood flow by approximately 25% at 2.5 mM and by > 100% at 10.0 mM. In isolated rat hearts, perfusion with 2.5 and 5.0 mM PBN for 60 min did not significantly affect global myocardial function, assessed as developed pressure, rate-pressure product, and positive and negative dP/dt. At the 10.0 mM concentration, however, these variables were significantly decreased after 30 min (developed pressure: -77% vs. controls; rate-pressure product: -84%; +dP/dt: -60%; -dP/dt: -70%); two out of five hearts stopped beating within 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)