Rationale Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes. Hyperglycemia, a hallmark of diabetes, is associated with increased vascular calcification as well as increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). Objective We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Methods and Results Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which also was associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase (OGA) that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or OGA knockdown, promoted calcification of primary mouse vascular smooth muscle cells (VSMC). Increased O-GlcNAcylation in diabetic arteries or in the OGA knockdown VSMC upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new O-sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced VSMC calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mTOR complex 2 to AKT, and subsequently blocked Runx2 transactivity and VSMC calcification. Conclusions O-GlcNAcylation of AKT at two new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes and uncovered a key molecular mechanism underlying O-GlcNAcylation-mediated activation of AKT.

Synopsis Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mitochondrial DNA (mtDNA) sequence variation contributes to disease susceptibility. In this study we present a novel animal model of mtDNA polymorphisms, the mitochondrial nuclear exchange mouse (MNX), in which the mtDNA from C3H/HeN mouse has been inserted onto the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harboring the C57/BL6J mtDNA generate more reactive oxygen species (ROS) and have a higher mitochondrial membrane potential relative to those having the C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the “mitochondrial paradigm” for the development of disease susceptibility, and show that the mtDNA modulates, cellular bioenergetics, mitochondrial reactive oxygen species generation and susceptibility to cardiac stress.

Abstract-c-kit, the transmembrane tyrosine kinase receptor for stem cell factor, is required for melanocyte and mast cell development, hematopoiesis, and differentiation of spermatogonial stem cells. We show here that in the heart, c-kit is expressed not only by cardiac stem cells but also by cardiomyocytes, commencing immediately after birth and terminating a few days later, coincident with the onset of cardiomyocyte terminal differentiation.

BackgroundThe clinical problem of a “pure volume overload” as in isolated mitral or aortic regurgitation currently has no documented medical therapy that attenuates collagen loss and the resultant left ventricular (LV) dilatation and failure. Here, we identify a potential mechanism related to upregulation of the kallikrein-kinin system in the volume overload of aortocaval fistula (ACF) in the rat.Methodology/Principal FindingsLV interstitial fluid (ISF) collection, hemodynamics, and echocardiography were performed in age-matched shams and 4 and 15 wk ACF rats. ACF rats had LV dilatation and a 2-fold increase in LV end-diastolic pressure, along with increases in LV ISF bradykinin, myocardial kallikrein and bradykinin type-2 receptor (BK2R) mRNA expression. Mast cell numbers were increased and interstitial collagen was decreased at 4 and 15 wk ACF, despite increases in LV ACE and chymase activities. Treatment with the kallikrein inhibitor aprotinin preserved interstitial collagen, prevented the increase in mast cells, and improved LV systolic function at 4 wk ACF. To establish a cause and effect between ISF bradykinin and mast cell-mediated collagen loss, direct LV interstitial bradykinin infusion in vivo for 24 hrs produced a 2-fold increase in mast cell numbers and a 30% decrease in interstitial collagen, which were prevented by BK2R antagonist. To further connect myocardial stretch with cellular kallikrein-kinin system upregulation, 24 hrs cyclic stretch of adult cardiomyocytes and fibroblasts produced increased kallikrein, BK2R mRNA expressions, bradykinin protein and gelatinase activity, which were all decreased by the kallikrein inhibitor-aprotinin.Conclusions/SignificanceA pure volume overload is associated with upregulation of the kallikrein-kinin system and ISF bradykinin, which mediates mast cell infiltration, extracellular matrix loss, and LV dysfunction–all of which are improved by kallikrein inhibition. The current investigation provides important new insights into future potential medical therapies for the volume overload of aortic and mitral regurgitation.

Volume overload (VO) caused by aortocaval fistula (ACF) is associated with oxidative/inflammatory stress. The resulting inflammation, matrix metalloproteinase (MMP) activation, and collagen degradation is thought to play a pivotal role in left ventricular (LV) dilatation and failure. Since mitochondria are also targets for inflammation and oxidative stress, we hypothesized that there would be bioenergetic dysfunction with acute VO. In Sprague-Dawley rats subjected to 24 hrs of ACF, there was a two-fold increase in LV pressure-volume area in vivo, consistent with increased LV myocardial oxygen usage and increased bioenergetic demand in cardiomyocytes. Isolated cardiomyocytes from ACF LVs demonstrated increased hydrogen peroxide and superoxide formation and increased MMP activity. Subsarcolemmal mitochondria (SSM) showed a 40% decrease in state 3 respiration and proteomic analysis of SSM demonstrated decreased levels of complexes I-V in ACF. Immunohistochemical analysis revealed disruption of the subsarcolemmal location of the SSM network in ACF. To test for a potential link between SSM dysfunction and loss of interstitial collagen, rats were treated with the MMP-inhibitor PD166793 prior to ACF. MMP-inhibitor preserved interstitial collagen, integrin-α5 and the SSM structural arrangement. In addition, the decrease in state 3 mitochondrial respiration with ACF was prevented by PD166793. These studies established an important interaction between degradation of interstitial collagen in acute VO and the disruption of SSM structure and function which could contribute to progression to heart failure.