MicroRNAs are posttranscriptional gene regulators that are differentially expressed during various diseases and have been implicated in the underlying pathogenesis. We report here that miR-199a is acutely downregulated in cardiac myocytes on a decline in oxygen tension. This reduction is required for the rapid upregulation of its target, hypoxia-inducible factor (Hif)-1α. Replenishing miR-199a during hypoxia inhibits Hif-1α expression and its stabilization of p53 and, thus, reduces apoptosis. On the other hand, knockdown of miR-199a during normoxia results in the upregulation of Hif-1α and Sirtuin (Sirt)1 and reproduces hypoxia preconditioning. Sirt1 is also a direct target of miR-199a and is responsible for downregulating prolyl hydroxylase 2, required for stabilization of Hif-1α. Thus, we conclude that miR-199a is a master regulator of a hypoxia-triggered pathway and can be exploited for preconditioning cells against hypoxic damage. In addition, the data demonstrate a functional link between 2 key molecules that regulate hypoxia preconditioning and longevity.

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...

Mammalian models of longevity are related primarily to caloric restriction and alterations in metabolism. We examined mice in which type 5 adenylyl cyclase (AC5) is knocked out (AC5 KO) and which are resistant to cardiac stress and have increased median lifespan of approximately 30%. AC5 KO mice are protected from reduced bone density and susceptibility to fractures of aging. Old AC5 KO mice are also protected from aging-induced cardiomyopathy, e.g., hypertrophy, apoptosis, fibrosis, and reduced cardiac function. Using a proteomic-based approach, we demonstrate a significant activation of the Raf/MEK/ERK signaling pathway and upregulation of cell protective molecules, including superoxide dismutase. Fibroblasts isolated from AC5 KO mice exhibited ERK-dependent resistance to oxidative stress. These results suggest that AC is a fundamentally important mechanism regulating lifespan and stress resistance.

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Rationale: Increased aortic stiffness, an important feature of many vascular diseases, eg, aging, hypertension, atherosclerosis, and aortic aneurysms, is assumed because of changes in extracellular matrix (ECM).Objective: We tested the hypothesis that the mechanisms also involve intrinsic stiffening of vascular smooth muscle cells (VSMCs). Methods and Results:

The posttranscriptional regulator, microRNA-21 (miR-21), is up-regulated in many forms of cancer, as well as during cardiac hypertrophic growth. To understand its role, we overexpressed it in cardiocytes where it revealed a unique type of cell-to-cell "linker" in the form of long slender outgrowths and branches. We subsequently confirmed that miR-21 directly targets and down-regulates the expression of Sprouty2 (SPRY2), an inhibitor of branching morphogenesis and neurite outgrowths. We found that beta-adrenergic receptor (betaAR) stimulation induces up-regulation of miR-21 and down-regulation of SPRY2 and is, likewise, associated with connecting cell branches. Knockdown of SPRY2 reproduced the branching morphology in cardiocytes, and vice versa, knockdown of miR-21 using a specific 'miRNA eraser' or overexpression of SPRY2 inhibited betaAR-induced cellular outgrowths. These structures enclose sarcomeres and connect adjacent cardiocytes through functional gap junctions. To determine how this aspect of miR-21 function translates in cancer cells, we knocked it down in colon cancer SW480 cells. This resulted in disappearance of their microvillus-like protrusions accompanied by SPRY2-dependent inhibition of cell migration. Thus, we propose that an increase in miR-21 enhances the formation of various types of cellular protrusions through directly targeting and down-regulating SPRY2.

Abstract-To seek evidence that the nonhuman primate arterial wall, as it ages in the absence of atherosclerosis, exhibits alterations in pathways that are involved in the pathogenesis of experimental atherosclerosis, we assessed aortic matrix metalloproteinase-2 (MMP-2) and its regulators, ie, membrane type-1 of matrix metalloproteinase (MT1-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), and the expression of angiotensin II (Ang II), angiotensinconverting enzyme (ACE), and chymase in young (6.4Ϯ0.7 years) and old (20.0Ϯ1.9 years) male monkeys. With advancing age, (1) the intimal thickness increased 3-fold and contained numerous vascular smooth muscle cells and matrix, but no inflammatory cells; (2) the intimal MMP-2 antibody-staining fraction increased by 80% (PϽ0.01); (3) in situ zymography showed that MMP-2 activity, mainly confined to the intima, increased 3-fold (PϽ0.01); (4) the MT1-MMP antibody-staining fraction increased by 150% (PϽ0.001), but the TIMP-2 antibody-staining fraction did not significantly change; (5) steady levels of the mRNA-staining fraction (via in situ hybridization) for MMP-2 increased 7-fold, for MT1-MMP increased 9-fold, and for TIMP-2 increased 2-fold (all PϽ0.001); and (6) intimal Ang II and ACE immunofluorescence were increased 5-fold and 5.6-fold, respectively, and colocalized with MMP-2. Thus, age-associated arterial remodeling and the development and progression of experimental atherosclerosis in young animals share common mechanisms, ie, MMP-2 activation and increased Ang II signaling. This might explain, in part, the dramatically exaggerated prevalence and severity of vascular diseases with aging.

Background-The adaptation of cardiac mass to hemodynamic overload requires an adaptation of protein turnover, ie, the balance between protein synthesis and degradation. We tested 2 hypotheses: (1) chronic left ventricular hypertrophy (LVH) activates the proteasome system of protein degradation, especially in the myocardium submitted to the highest wall stress, ie, the subendocardium, and (2) the proteasome system is required for the development of LVH. Methods and Results-Gene and protein expression of proteasome subunits and proteasome activity were measured separately from left ventricular subendocardium and subepicardium, right ventricle, and peripheral tissues in a canine model of severe, chronic (2 years) LVH induced by aortic banding and then were compared with controls. Both gene and protein expressions of proteasome subunits were increased in LVH versus control (PϽ0.05), which was accompanied by a significant (PϽ0.05) increase in proteasome activity. Posttranslational modification of the proteasome was also detected by 2-dimensional gel electrophoresis. These changes were found specifically in left ventricular subendocardium but not in left ventricular subepicardium, right ventricle, or noncardiac tissues from the same animals. In a mouse model of chronic pressure overload, a 50% increase in heart mass and a 2-fold increase in proteasome activity (both PϽ0.05 versus sham) were induced. In that model, the proteasome inhibitor epoxomicin completely prevented LVH while blocking proteasome activation. Conclusions-The increase in proteasome expression and activity found during chronic pressure overload in myocardium submitted to higher stress is also required for the establishment of LVH. Key Words: heart diseases Ⅲ hypertrophy Ⅲ physiology Ⅲ pressure Ⅲ stress Ⅲ proteins L eft ventricular hypertrophy (LVH) is a key compensatory mechanism in response to pressure or volume overload that involves alterations in the regulation of signal transduction pathways, transcription factors, excitation-contraction coupling, contractile proteins, and energy metabolism. One key element of cardiac hypertrophy is an adaptation in protein turnover. Protein turnover refers to protein synthesis and degradation, and both mechanisms are activated by increased cardiac workload. 1,2 Although multiple studies have addressed the activation of protein synthesis during the acute phase of LVH that follows aortic banding, the mechanisms controlling protein degradation in the hypertrophied myocardium, especially over the long term, remain largely unknown. A key mechanism involved in protein degradation is the ubiquitin/proteasome system (UPS), 3 which is known to be an important mechanism mediating muscle atrophy. 4,5 Proteolytic substrates are ligated to multiple ubiquitin (Ub) moieties that are assembled into a chain that binds the proteasome with high affinity. The 26S proteasome contains multiple subunits in the regulatory (19S) particle that can bind multiubiquitinated (multi-Ub) proteins. 6,7 The composition of the proteasome is highl...