Abstract-Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-␤-D-arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1 ϩ/Ϫ mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1-dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. (Circ Res. 2007;100:914-922.) Key Words: autophagy Ⅲ AMP-activated protein kinase (AMPK) Ⅲ beclin 1 Ⅲ ischemia/reperfusion A utophagy is an intracellular bulk degradation process whereby cytosolic, long-lived proteins and organelles are degraded and recycled. 1 Autophagy occurs at basal levels but can be further induced by stresses, such as nutrient depletion. 2 Autolysosomal degradation of membrane lipids and proteins generates free fatty acids and amino acids, which can be reused to maintain mitochondrial ATP production and protein synthesis, and promote cell survival. Disruption of this pathway prevents cell survival in diverse organisms. 2 Interestingly, autophagy also promotes programmed cell death in some circumstances. 3,4 Thus, autophagy has a dual role in cell survival, although, in cardiac myocytes, it remains to be elucidated whether autophagy is required for survival, and is thereby salutary, or whether it mediates cell death, and is detrimental during pathologically relevant stresses, such as ischemia and reperfusion.The mTOR (mammalian target of rapamycin) pathway is a key regulator of cell growth and proliferation and integrates signals regarding nutrie...

We tested the hypothesis that chronically ischemic (IS) myocardium induces autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which could protect against the consequences of further ischemia. Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis (30% reduction in baseline coronary flow followed by reperfusion every 12 h) with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by Ϸ37%. Using a nonbiased proteomic approach combining 2D gel electrophoresis with in-gel proteolysis, peptide mapping by MS, and sequence database searches for protein identification, we demonstrated increased expression of cathepsin D, a protein known to mediate autophagy. Additional autophagic proteins, cathepsin B, heat shock cognate protein Hsc73 (a key protein marker for chaperone-mediated autophagy), beclin 1 (a mammalian autophagy gene), and the processed form of microtubule-associated protein 1 light chain 3 (a marker for autophagosomes), were also increased. These changes, not evident after one episode, began to appear after two or three episodes and were most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically IS myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. Immunohistochemistry staining for cathepsin B was more intense in areas where apoptosis was absent. Thus, autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited.proteomics ͉ lyposomal proteins ͉ apoptosis ͉ hibernating myocardium ͉ myocardial protection A utophagy is a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins and has been studied extensively in lower organisms such as yeast, Caenorhabditis elegans, and Drosophila (1-4). Autophagy has been suggested to be an essential function for cell homeostasis and cell defense and adaptation to an adverse environment (1, 2, 5). Autophagy is typically activated by starvation, when the cytoplasmic proteins or organelles are delivered to the lysosome and degraded (1-4). In autophagy, cytoplasmic proteins or dysfunctional organelles are sequestrated in a doublemembrane-bound vesicle, termed autophagosome, delivered to the lysosome by fusion, and then degraded. Autophagy allows the cell not only to recycle amino acids but also to remove damaged organelles, thereby eliminating oxidative stress and allowing cellular remodeling for survival (2, 6). In fact, autophagy is a cellular mechanism essential for dauer development and lifespan extension in C. elegans (1). It can also prevent accumulation of misfolded and aggregated proteins in Parkinson's, Huntington's, and Alzheimer's disease...

Abstract-Osteopontin (OPN) is upregulated in several experimental models of cardiac fibrosis and remodeling. However, its direct effects remain unclear. We examined the hypothesis that OPN is important for the development of cardiac fibrosis and remodeling. Moreover, we examined whether the inhibitory effect of eplerenone (Ep), a novel aldosterone receptor antagonist, was mediated through the inhibition of OPN expression against cardiac fibrosis and remodeling. Wild-type (WT) and OPN-deficient mice were treated with angiotensin II (Ang II) for 4 weeks. WT mice receiving Ang II were divided into 2 groups: a control group and an Ep treatment group. Ang II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in WT mice. Ep treatment and OPN deficiency could reduce the Ang II-induced elevation of blood pressure and ameliorate the development of cardiac fibrosis, whereas Ep-only treatment abolished the development of cardiac hypertrophy. Most compelling, the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and subsequent left ventricular dilatation in Ang II-treated OPN-deficient mice. These results suggest that OPN has a pivotal role in the development of Ang II-induced cardiac fibrosis and remodeling. Moreover, the effect of Ep on the prevention of cardiac fibrosis, but not cardiac hypertrophy, might be partially mediated through the inhibition of OPN expression. Osteopontin (OPN) is reported to be involved in the process of Ang II-induced fibrosis. 3 Furthermore, OPN can interact with various extracellular matrices, including fibronectin and collagen, suggesting its possible role in matrix organization and stability. 4 Recently, it was shown that OPN expression in heart was associated with the development of heart failure. 5 Moreover, in a murine model of myocardial infarction, OPN deficiency caused exaggeration of left ventricular (LV) dilation and reduction of collagen deposition compared with wild-type (WT) mice. 6 These results suggest that OPN has a pivotal role in cardiac fibrosis and cardiac remodeling.More recently, an important link was suggested in Ang II-induced cardiac fibrosis between OPN and Ald. Ang II induced inflammatory damage in coronary arteries and OPN expression, and eplerenone (Ep), a novel Ald receptor antagonist, could inhibit the OPN expression and ameliorate the Ang II-induced inflammatory damage to coronary arteries. 7 These results suggested that (1) OPN-mediated vascular inflammation might be part of the mechanism by which the renin-angiotensin-aldosterone system participates in the development of cardiac fibrosis and (2) the effect of Ep on the inhibition of vascular inflammation might be modulated by suppressing OPN expression.To investigate whether OPN plays a pivotal role in cardiac fibrosis and remodeling, we treated OPN-deficient (OPN Ϫ/Ϫ ) mice with Ang II and compared them with WT mice treated with Ang II alone or with Ang II and Ep. Herein, we report the role of OPN and the relationship between OPN and Ald ...

Objective-Osteopontin (OPN), a noncollagenous adhesive protein, is implicated in atherosclerosis, in which macrophages within atherosclerotic plaques express OPN. However, it is not known whether the elevated OPN expression is a cause or result of atherosclerosis. Methods and Results-We generated mice that lacked OPN and crossed them with apolipoprotein (apo) E-deficient mice and analyzed these mice with a mixed C57BL/6ϫ129 background after 36 weeks on a normal chow diet. In female mice, OP ϩ/Ϫ E Ϫ/Ϫ and OP Ϫ/Ϫ E Ϫ/Ϫ mice had significantly smaller atherosclerotic and inflammatory lesions compared with OP ϩ/ϩ E Ϫ/Ϫ mice, and that was reflected by smaller area of MOMA-2-positive staining. In male mice, however, there was no significant difference in the atherosclerosis lesion areas among 3 genotypes. In both OP Ϫ/Ϫ E Ϫ/Ϫ and OP ϩ/ϩ E Ϫ/Ϫ mice, typical atherosclerotic lesions were detected, which include necrotic core, foamy cell collections, and cholesterol clefts. However, we found that vascular mineral-deposited areas in 60-week-old male OP Ϫ/Ϫ E Ϫ/Ϫ mice were significantly increased compared with those in OP ϩ/ϩ E Ϫ/Ϫ male mice. Key Words: osteopontin Ⅲ atherosclerosis Ⅲ macrophage Ⅲ calcification Ⅲ lipid metabolism A therosclerosis is characterized as a chronic inflammatory process of the vessel wall. Atherosclerosis is initiated by the infiltration of monocytes and T-lymphocytes into activated endothelium, followed by their migration into the intima and subsequent lipid accumulation within macrophages. In late stages of atherosclerosis, calcification is a common advanced complication. Osteopontin (OPN), a noncollagenous adhesive protein, was first found at sites of dystrophic calcification and is synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. 1 The expression of OPN protein was detected in not only macrophages but also vascular smooth muscle cells within atherosclerotic lesion. [1][2][3][4][5] In addition, it was shown that vascular smooth muscle cells during the proliferative and migratory phase, but not the quiescent and contractile phase, expressed OPN in a model of balloon catheter injury of rat carotid artery. 2 More importantly, Liaw et al 6 reported that neutralizing antibodies directed against OPN inhibited rat carotid neointimal thickening after endothelial denudation. Taken together, these results suggested that OPN can play a pivotal role in the early stage of atherosclerosis, including proliferation and migration of smooth muscle cells as well as at the late stage of atherosclerosis, characterized by calcified atheromatous plaque formation. Conclusions-TheseTo more directly address the question of whether OPN initiates the development of atherosclerotic lesion and plays a role in calcification, we took advantage of OPN-deficient mice we had recently generated. 7 We crossed them with apolipoprotein (apo) E-deficient mice and made OPN and apoE double-deficient mice. In mice deficient for OPN gene expression, OPN mRNA was not detected in any organs. ...