Chronic -Adrenergic Receptor Stimulation Induces Cardiac Apoptosis and Aggravates Myocardial Ischemia/Reperfusion Injury by Provoking Inducible Nitric-Oxide Synthase-Mediated Nitrative Stress

The present study provides evidence that inducible nitric-oxide synthase (iNOS)-mediated nitrative stress plays a pivotal role in chronic -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 (iNOSknockout 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 -AR stimulation upregulates 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. Catecholamine release secondary to activation of the sympathetic nervous system is a key neuromodulator of cardiovascular, metabolic, and other physiological functions in humans. However, a prolonged release or sudden surge of catecholamine, as seen in conditions such as major surgery, trauma, sepsis, and congestive heart failure, can profoundly increase the patient’s risk for cardiac complications, including cardiac arrhythmias, myocardial ischemia (MI) or/and infarction, and sudden cardiac death (Bristow, 2000; Lefkowitz et al., 2000). Both apoptotic and necrotic modes of cell death have been proposed as the mechanism underlying these cardiac complications. Many studies have addressed how catecholamine, especially -adrenergic receptor (AR) stimulation, induces cardiac apoptosis or/and necrosis (Colucci et al., 2000; Remondion et al., 2003). Recent studies also found that in failing myocardium, increased inducible nitric-oxide synthase (iNOS) activity contributes to attenuate -AR-mediated inotropic effect (Funakoshi et al., 2002; Gealekman et al., 2002). However, the role of iNOS in chronic -AR stimulation-induced cardiac damage remains to be investigated. Nitric oxide (NO), a reactive nitrogen species (RNS), is produced by three isoforms of NOS: endothelial NOS (eNOS), neuronal NOS (nNOS), and iNOS. All of them are present in This study was supported in part by National Institutes of Health Grant RO1 HL-63828 (X.L.M.) and a FAER (Foundation for Anesthesia Education and Research) grant (J.Z.S.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.102160. ABBREVIATIONS: MI, myocardial ischemia; AR, adrenergic receptor; iNOS, inducible nitric-oxide synthase; NO, nitric oxide; RNS, reactive nitrogen species; eNOS, endothelial nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; R, reperfusion; ISO, isoproterenol; 1400W, N-(3-(aminomethyl) benzyl)acetamidine; KO, knockout; PCR, polymerase chain reaction; LV, left ventricular; ELISA, enzyme-linked immunosorbent assay; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CRP, C-reactive protein; TTC, 2,3,5-triphenyltetrazolium chloride; AAR, area at risk; IL, interleukin. 0022-3565/06/3182-469–475$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 102160/3119014 JPET 318:469–475, 2006 Printed in U.S.A. 469 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from the heart (Kelly et al., 1996). Unlike eNOS and nNOS, iNOS has not been found to be constitutively present in the normal, nonaged, adult heart, but it can be induced by proinflammatory substances such as cytokines and lipopolysaccharide or conditions such as stroke, trauma, infection, and a variety of cardiovascular diseases (Haywood et al., 1996; Kelly et al., 1996). Once induced, iNOS isoform is capable of producing large amounts of NO at 100 times greater than normal ( 1 M versus 10 nM) by resident cardiac cells and activated immune cells that infiltrate the injured heart (Nathan, 1997; Mungrue et al., 2002; Ferdinandy and Schulz, 2003). Such high levels of NO are recognized as a mediator and regulator of inflammatory responses. Recently, our studies found that acute -AR stimulation in aging ischemic heart triggered a marked increase in NO production, generated toxic peroxynitrite, activated apoptosis, and eventually caused cardiac dysfunction and myocardial injury (Li et al., 2006). However, whether the induction or/and expression of iNOS is beneficial or deleterious over the long term, particularly its effects on myocardial apoptosis and MI/reperfusion (R) injury, remains