The major hypothesis to be tested in this proposal is that, ?Reducing the excess addition of ?-N- acetylglucosamine (O-GlcNAc) to regulatory factors present in cardiac fibroblasts ameliorates diabetes mellitus induced myocardial fibrosis?. Given the older average age of the Veteran population, about 25% suffer from type 2 diabetes mellitus (DM2). A large number of these patients will develop DM2 induced cardiac fibrosis, which adversely impacts diastolic function and frequently leads to the development of heart failure. Excess O- GlcNAc modification of proteins is known to occur with aging and most notably in the setting of DM2. We have demonstrated that excess O-GlcNAcylation of cardiac fibroblast (CF) proteins is associated with the enhanced production of collagens. As tissue fibrosis (e.g. glomerulosclerosis) is a prominent feature of DM2 our research findings may also have broader implications as a strategy to ameliorate excess collagen production by fibroblasts present in other organs. The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ?-N-acetylglucosamine is a highly dynamic and ubiquitous protein modification that is secondary to the action of ?-N- acetylglucosaminyltransferase (OGT). Conversely, the removal of O-GlcNAc is mediated by N- acetylglucosaminidase (O-GlcNAcase). Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. We demonstrated that in high glucose treated CF, the nuclear transcription factor Sp1 and arginase evidence excess O-GlcNAcylation. Both of these proteins are intricately associated with stimulating the production of collagens. Expression in CF of an adenovirus coding for O-GlcNAcase, decreased Sp1 and arginase O-GlcNAcylation and restores high glucose- induced excess collagen production back to normal levels. However, no studies have identified which specific amino acid residues can be modified by O-GlcNAcylation and how they alter Sp1 and arginase function. Furthermore, these observations have not been evidenced in the in vivo setting and linked with changes in cardiac structure and function. Given these facts and the preliminary data we have generated, we propose to examine the following specific aims:
Aim 1. To identify the development of myocardial fibrosis, diastolic dysfunction, Sp1 and arginase I residue modification in an aged model of DM2.
Aim 2. To characterize HG induced CF amino acid residue O-GlcNAc modification of Sp1 and arginase I and its functional implications.
Aim 3. To characterize the capacity of altered O-GlcNAcase activity to modify DM2 or HG induced alterations in Sp1 and arginase I residue modification, fibroblast/myofibroblast phenotype, cardiac structure and function.
Type 2 diabetes mellitus and heart disease are an increasing serious health concern in the Veteran population in particular, as the population ages and are exposed to risk factors such as the use of antipsychotic drugs. Nearly 70% of male veterans are older than 55 years of age and the majority of female veterans are over 45 years of age (National Center for Veterans Analysis and Statistics report 2011). The current proposal aims to address the molecular mechanism involved in the development of cardiac fibrosis in diabetic and non-diabetic patients and the role played by the modification of proteins by glucose. The work described in this proposal will also identify possible novel therapeutic strategies that may counter the adverse effects of diabetes on the heart.