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.

Type 2 diabetes mellitus (T2DM) affects a large population worldwide. T2DM is a complex heterogeneous group of metabolic disorders including hyperglycemia and impaired insulin action and/or insulin secretion. T2DM causes dysfunctions in multiple organs or tissues. Current theories of T2DM include a defect in insulin-mediated glucose uptake in muscle, a dysfunction of the pancreatic b-cells, a disruption of secretory function of adipocytes, and an impaired insulin action in liver. The etiology of human T2DM is multifactorial, with genetic background and physical inactivity as two critical components.The pathogenesis of T2DM is not fully understood. Animal models of T2DM have been proved to be useful to study the pathogenesis of, and to find a new therapy for, the disease. Although different animal models share similar characteristics, each mimics a specific aspect of genetic, endocrine, metabolic, and morphologic changes that occur in human T2DM. The purpose of this review is to provide the recent progress and current theories in T2DM and to summarize animal models for studying the pathogenesis of the disease.

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.

A high-fiber dietary pattern and subsequent consistent production of SCFAs and healthy gut microbiota are associated with a reduced risk of A-CRA. This trial was registered at www.chictr.org as ChiCTR-TRC-00000123.

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...

Sepsis is a systemic inflammatory response to infection that causes severe neurological complications. Previous studies have suggested that melatonin is protective during sepsis. Additionally, silent information regulator 1 (SIRT1) was reported to be beneficial in sepsis. However, the role of SIRT1 signaling in the protective effect of melatonin against septic encephalopathy remains unclear. This study aimed to investigate the role of SIRT1 in the protective effect of melatonin. EX527, a SIRT1 inhibitor, was used to reveal the role of SIRT1 in melatonin's action. Cecal ligation and puncture or sham operation was performed in male C57BL/6J mice. Melatonin was administrated intraperitoneally (30 mg/kg). The survival rate of mice was recorded for the 7-day period following the sham or CLP operation. The blood-brain barrier (BBB) integrity, brain water content, levels of inflammatory cytokines (TNF-α, IL-1β, and HMGB1), and the level of oxidative stress (superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)) and apoptosis were assessed. The expression of SIRT1, Ac-FoxO1, Ac-p53, Ac-NF-κB, Bcl-2, and Bax was detected by Western blot. The results suggested that melatonin improved survival rate, attenuated brain edema and neuronal apoptosis, and preserved BBB integrity. Melatonin decreased the production of TNF-α, IL-1β, and HMGB1. Melatonin increased the activity of SOD and CAT and decreased the MDA production. Additionally, melatonin upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of Ac-FoxO1, Ac-p53, Ac-NF-κB, and Bax. However, the protective effects of melatonin were abolished by EX527. In conclusion, our results demonstrate that melatonin attenuates sepsis-induced brain injury via SIRT1 signaling activation.

Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by nearly 45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity (PWV) and blood pressure (BP) in Klotho-haplodeficient (KL+/−) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL+/− mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/day, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in KL+/− mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase alpha (AMPKα) and endothelial nitric oxide synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension.