Urocortin (UCN) is a peptide related to hypothalamic corticotrophin-releasing hormone and binds with high affinity to corticotrophin-releasing hormone receptor-2␤, which is expressed in the heart. In this study, we report that UCN prevented cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia. UCN-mediated cell survival was measured by trypan blue exclusion, 3-OH end labeling of DNA (TUNEL), annexin V, and fluorescence-activated cell sorting. To explore the mechanisms that could be responsible for this effect, we investigated the involvement of MAPK-dependent pathways. UCN caused rapid phosphorylation of ERK1/2-p42/ 44, and PD98059, which blocks the MEK1-ERK1/2-p42/44 cascade, also inhibited the survival-promoting effect of UCN. Most important, UCN reduced damage in isolated rat hearts ex vivo subjected to regional ischemia/reperfusion, with the protective effect being observed when UCN was given either prior to ischemia or at the time of reperfusion after ischemia. This suggests a novel function of UCN as a cardioprotective agent that could act when given after ischemia, at reperfusion. Urocortin (UCN)1 is a peptide related to the hypothalamic hormone corticotrophin-releasing factor (CRF), the central mediator of the hypothalamic-pituitary-adrenal axis and stress response in mammals (1-3). UCN and CRF share 45% homology at the amino acid level, and both are synthesized as precursors, which are subsequently processed to the mature biologically active peptides (in the case of UCN, a 40-amino acid molecule) (4, 5). Although UCN was originally identified in restricted areas of the brain, it has also been found in the placenta, lymphocytes, and heart (6 -9).The CRF family of peptides bind two types of CRF receptors, CRF-R1 and CRF-R2. CRF-R2 exists in three alternative splice variant forms, CRF-R2␣, CRF-R2␤, and CRF-R2␥ (10 -12); and CRF-R2 binds UCN with a higher affinity than CRF both in ligand binding studies (4) and as shown by the effects of ligand on intracellular cAMP (13). In contrast, the R1 receptors show little ligand selectivity for UCN versus CRF. R2 receptors are the only type of CRF receptor found in the heart: the ␣ form in man (14) and the ␤ form in the rat (15). The CRF family of peptides have been shown to stimulate adenylate cyclase activity in cardiac myocytes (16), and changes in CRF-R2 expression have been reported in the hearts of spontaneously hypertensive rats (17).The coincident expression of CRF-R2 receptors with their preferred UCN ligand in the heart suggests that UCN may have physiological cardiac properties. Indeed UCN, but not CRF, induces a dose-dependent increase in heart rate, cardiac output, and coronary blood flow (18). Moreover, cardiac CRF-R2␤ expression is modulated by endotoxin, a potent inducer of cardiovascular dysregulation, further suggesting a possible link between UCN and the cardiovascular response to stress (19). Indeed, in previous studies, we have...

We show here that exposure of cardiac cells to simulated ischemia results in apoptosis and is accompanied by phosphorylation and increased expression and transcriptional activity of STAT-1. Similarly, interferon-␥, which is known to induce STAT-1 activation, also induced apoptosis in cardiac cells. STAT-1-transfected cells were more susceptible to ischemia-induced cell death than cells transfected with a control plasmid lacking the STAT-1 coding sequence. Furthermore, an antisense STAT-1 vector reduced both ischemia-and overexpressed STAT-1-induced cell death in cardiac cells. Both STAT-1 overexpression and interferon-␥ treatment or exposure to ischemia activated the promoter of the pro-apoptotic caspase-1 gene in cardiomyocytes. Finally, ischemia/reperfusion also induced STAT-1 activation and caspase-1 processing in ventricular myocytes in the intact heart ex vivo. Immunofluorescent staining demonstrated an increase in STAT-1-positive staining in cardiomyocytes in response to ischemia/reperfusion that co-localized with terminal deoxynucleotidyl transferase dVTP nick end-labeling-positive apoptotic cells. These results suggest that STAT-1 plays a critical role in the regulation of ischemia/reperfusion-induced apoptosis in cardiac cells, acting at least in part via a caspase-1 activation-dependent pathway.Loss of cardiomyocytes by programmed cell death (apoptosis) is an important mechanism in the development of cardiac failure during injury due to ischemia/reperfusion and myocardial infarction (1, 2). Recent studies have indicated that apoptotic death occurs in cardiac cells exposed to a variety of damaging stimuli both in vitro and in the intact heart in vivo (3-6). Thus, cardiac cells exposed to a hypoxic/ischemic insult followed by reperfusion undergo apoptotic cell death in vitro (3, 6). Similarly, apoptotic cell death is also observed in the intact heart following ischemia in vivo (4, 5). Despite the convincing evidence that apoptosis occurs, the mechanism and signaling pathway that leads to hypoxic/ischemic stimuli resulting in apoptosis in cardiac cells is as yet unknown. However, as in other cell types, caspases have been implicated in apoptotic cell death in cardiomyocytes (7). For example, the infarct size following ischemia/reperfusion of the intact heart in vivo can be reduced by priming animals with the nonspecific caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, directly demonstrating the role of caspases in mediating cell death in cardiac cells (8).Cytokines and growth factors are known to modulate growth, differentiation, and death in many cell types. For example, interferons (IFNs) 1 have been shown to trigger cell cycle arrest and death in non-cardiac cells (9, 10), whereas many interleukins stimulate growth and protect cells from apoptosis (11, 12). These pro-and anti-apoptotic effects are mediated, at least in part, by signaling through a family of transcription factors called STATs. Six STATs have been cloned, some of which exist in different isomeric forms, and all shar...