In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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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...

In the very early stages of reperfusion, apoptosis is first seen in the endothelial cells from small coronary vessels. The radial spread of apoptosis to surrounding cardiac myocytes suggests that reperfusion induces the release of soluble pro-apoptotic mediators from endothelial cells that promote myocyte apoptosis.

The STAT-1 transcription factor has been implicated as a tumor suppressor by virtue of its ability to inhibit cell growth and promoting apoptosis. However, the mechanisms by which STAT-1 mediates these effects remain unclear. Using human and mouse STAT-1-deficient cells, we show here that STAT-1 is required for optimal DNA damage-induced apoptosis. The basal level of the p53 inhibitor Mdm2 is increased in STAT-1(؊/؊) cells, suggesting that STAT-1 is a negative regulator of Mdm2 expression. Correspondingly, both basal p53 levels, and those induced by DNA damage were lower in STAT-1(؊/؊) cells. In agreement with this lower p53 response to DNA damage in cells lacking STAT-1, the induction of p53 responsive genes, such as Bax, Noxa, and Fas, was reduced in STAT-1-deficient cells. Conversely, STAT-1 overexpression enhances transcription of these genes, an effect that is abolished if the p53 response element in their promoters is mutated. Moreover, STAT-1 interacts directly with p53, an association, which is enhanced following DNA damage. Therefore, in addition to negatively regulating Mdm2, STAT-1 also acts as a coactivator for p53. Hence STAT-1 is another member of a growing family of protein partners able to modulate the p53-activated apoptotic pathway.

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...

Previously we reported that ischemia results in apoptosis and is accompanied by phosphorylation on Tyr-701 and increased expression and transcriptional activity of the signal transducer and activator of transcription-1 (STAT-1). In the present study, we show that exposure of cardiomyocytes to ischemia induced the phosphorylation of STAT-1 at another site, Ser-727. Moreover, STAT-1 is critical for the induction of Fas receptor and Fas ligand expression by ischemia/reperfusion (I/R). Transcriptional activation of Fas and

We have previously demonstrated that STAT-1 plays a critical role in promoting apoptotic cell death in cardiac myocytes following ischemia/reperfusion (I/R) injury. Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has recently been reported to inhibit STAT-1 activity in noncardiac cells. In the present study, we have assessed the protective effects of EGCG and green tea extract (GTE) infusion on both cultures of cardiac myocytes and the isolated rat heart. EGCG reduced STAT-1 phosphorylation and protected cardiac myocytes against I/R-induced apoptotic cell death. Moreover, EGCG reduced the expression of a known STAT-1 pro-apoptotic target gene, Fas receptor. More interestingly, oral administration of GTE as well as EGCG infusion limited the extent of infarct size and attenuated the magnitude of myocyte apoptosis in the isolated rat heart exposed to I/R injury. This reduction cell death was associated with improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart. This is the first report to show that consumption of green tea is able to mediate cardioprotection and enhance cardiac function during I/R injury. Because GTE-mediated cardioprotection is achieved, at least in part, through inhibition of STAT-1 activity, we may postulate that a similar action can be implemented in the clinical setting to minimize STAT-1 activation levels in patients with acute coronary artery disease (CAD).