Matrix metalloproteinase (MMP)-2 belongs to a family of zinc-dependent proteases which are best known for their ability to proteolyse extracellular matrix proteins throughout the body, including the cardiovascular system. Increased MMP-2 activity has been demonstrated in myocardial ischaemia and reperfusion injury and the progression to congestive heart failure, with most evidence to date for its role in cardiac remodelling. Recent evidence, however, shows that MMP-2 also co-localizes with and proteolyses specific protein targets within the cardiomyocyte to cause acute, reversible contractile dysfunction, challenging the conventional wisdom on the ‘extracellular matrix only’ actions of this enzyme. In this review, we discuss the recent upsurge in MMP-2 research with regards to its activation by non-proteolytic pathways in the setting of enhanced oxidative stress in the heart. We will focus on the consequences of intracellular actions of MMP-2 within the cardiomyocyte and its regulation at several levels including its expression, post-translational modifications, and regulation by endogenous tissue inhibitors of metalloproteinases, caveolin, and small molecule MMP inhibitors. MMP-2 is emerging as an important signalling protease implicated in the proteolytic regulation of various intracellular proteins in myocardial oxidative stress injury.
BACKGROUND AND DESIGN: Adiponectin is an adipokine secreted primarily from adipose tissue that can inﬂuence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance. METHODS AND RESULTS: The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed signiﬁcantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins. CONCLUSION: Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity
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Aims Matrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3b is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3b is a MMP-2 substrate as a result of oxidative stress. Methods and results MMP-2 and GSK-3b were incubated and the cleavage fragments were identified by immunoblotting and silver stain. The intact protein and its primary cleavage fragment were subjected to trypsin digestion and the resultant peptides were analysed by LC–MS/MS. GSK-3b kinase activity was measured using a peptide substrate and [g-32P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H2O2 and the levels and activities of MMP-2 and GSK-3b were measured. Incubation of 47 kDa GSK-3b with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3b as seen by appearance of an 30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3b lacking the N-terminal region. GSK-3b kinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H2O2 challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3b, and significantly increased GSK-3b kinase activity. Both the loss of intact GSK-3b and increase in its kinase activity were reduced with MMP inhibitors. MMP-2 pulldown assays in H9c2 cell lysates showed the association of MMP-2 with GSK-3b. Conclusion GSK-3b may be a target of MMP-2 and its cleavage by MMP-2 enhances its kinase activity. MMP-2 may cleave off the N-terminal of GSK-3b where the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3b kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.