Maternal Diabetes as It Affects Extracellular Matrix Deposition in the Embryonic Mouse Heart

mouse;cardiogenesis;embryo;diabetes

Bullock, Nia Joyner ; Dr. John Gadsby, Committee Member,Dr. Brenda Alston-Mills, Committee Chair,Dr. Brenda J.Brizuela, Committee Member,Dr. Betty L. Black, Committee Member,Bullock, Nia Joyner ; Dr. John Gadsby ; Committee Member ; Dr. Brenda Alston-Mills ; Committee Chair ; Dr. Brenda J.Brizuela ; Committee Member ; Dr. Betty L. Black ; Committee Member

University:North Carolina State University

关键词: mouse;    cardiogenesis;    embryo;    diabetes;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/3312/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Fibrosis is a condition common to adults suffering from diabetes mellitus (DM), characterized by excessive production of extracellular matrix (ECM) proteins in the ECM of various organs and vasculature.Those with DM suffer from a wide range of health conditions such as peripheral nerve disorders, retinopathy, vascular disorders, nephropathy, and heart disease. Many of these effected tissues and organs develop fibrosis as a result of DM, which may eventually cause tissue dysfunction.As the ECM plays an important role in adult organ and tissue function, it also functions in cardiogenesis by supporting cell migration in early heart development and formation of septum and valves of the heart.Gestational diabetes mellitus (GDM) is a metabolic disorder that is characterized by the inability to properly metabolize glucose using insulin, in which onset of the disease occurs during pregnancy.Approximately 4% of pregnant women suffer from GDM and it is essential to treat and control GDM to lessen the risk of diabetic embryopathy.Common manifestations of diabetic embryopathy are malformations in the developing heart, spine, brain and lungs. Heart malformations are the most common defects observed in the offspring of diabetic mothers, and they include, but are not limited to, atrial and ventricular septal defects, outflow tract defects, cardiac valve defects, and increased interventricular septal thickness.As stated previously, the ECM supports heart valve and septal formation during cardiogenesis, women suffering from GDM are more likely to have offspring with heart defects, and adults with DM suffer from fibrotic conditions which affect cardiac function.Therefore, the goal of this dissertation is to model GDM in the pregnant mouse, explore the effects of GDM on glucose transport in the embryonic heart, examine cardiac ECM protein expression and reveal mechanisms by which alterations in cardiac ECM deposition may occur.We propose that GDM will alter the transport of glucose into cardiac cells, causing an alteration in the expression of cardiac ECM proteins, potentially leading to a fibrotic condition in these offspring which may eventually influence cardiac function in adulthood. In order to examine the effect of GDM on cardiac glucose transporter-1 (Glut-1) expression in the pregnant mouse, we induced hyperglycemia for 6 or 12 hours in vivo using a 6 g/kg intraperitoneal (IP) injection of D-glucose in saline on gestational day (gd) 9.5.Total heart protein was measured using a Bradford assay and cardiac Glut-1 protein and gene expression was measured using immunohistochemistry (IHC), SDS-PAGE Western analysis, and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).In efforts to examine durations of GDM for 24 hours, 48 hours, 6 days or 9 days in the pregnant mouse, we administered an IP injection of 250 mg/kg of streptozotocin (STZ) in citrate buffer on gd 7.5, rendering the pregnant mouse diabetic by gd 9.5.In efforts to discover whether changes in glucose transport induced changes in key regulators of the ECM and ECM expression we examined the cardiac protein and gene expression of transforming growth factor beta-1 (TGFβ-1), connective tissue growth factor (CTGF), and fibronectin (FN) using IHC, SDS-PAGE Western analysis, and Real time RT-PCR.Finally, using our STZ-induced GDM model, we explored other mechanisms that support ECM protein deposition such as cardiac expression of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) using IHC and Real time RT-PCR.In addition to our in vivo model, we used an in vitro model which was comprised of a primary embryonic heart cell culture system that exposed the embryonic heart cells to normoglycemic and hyperglycemic conditions.We then compared the production of MMPs, TIMPs and FN in the media of cells exposed to hyperglycemic and normoglycemic conditions, using ELISA and Real-Time PCR. Immediately following 6 and 12 hours of hyperglycemia in vivo, Glut-1 protein and gene expression were increased in the embryonic mouse heart.Cardiac Glut-1 protein and gene expression were decreased following 12 hours of hyperglycemia and 12 hours of normoglycemia while total cardiac protein was decreased following 6 and 12 hours of hyperglycemia and 18 or 12 hours of normoglycemia, respectively.TGFβ-1 and CTGF protein and gene expression were collectively increased at 24 hours in the embryonic mouse heart.FN protein expression was increased at 24 hours and remained elevated until the end of gestation, while gene expression was increased at 24 hours but returned to normal and remained unchanged throughout the remainder of gestation.STZ-induced GDM produced increases in Collagen IV and V, and laminin cardiac protein expression at 24 hours and these ECM proteins remained elevated until the end of gestation.STZ-induced GDM also produced decreases in cardiac MMP-2 and increases in cardiac TIMP1-2, and 3 protein and gene expression.ELISA and Real Time-PCR results from embryonic heart cell culture media and embryonic heart cells respectively, demonstrated decreases in MMP-2 protein and gene expression and increases in TIMP-1 protein and gene expression.Despite the hyperglycemic condition, Glut-1 protein and gene expression was increased in the embryonic mouse heart immediately following 6 and 12 hours of hyperglycemia, which suggests an increase in glucose uptake in embryonic heart cells.The compensatory measure of decreasing Glut-1 in a hyperglycemic environment did not occur until 12 hours after the hyperglycemic insult has passed.The brief increase observed in TGFβ-1 following 24 hours of STZ-induced GDM, suggest the ability of increased glucose uptake to induce cardiac TGFβ-1 expression.TGFβ-1 would in turn induce cardiac CTGF, all of which are key mediators in pathways leading to increased ECM deposition, which was observed by increases in cardiac FN expression.STZ-induced GDM caused a continuous increase in cardiac FN protein expression throughout the treatment period, while gene expression was up-regulated briefly and then returned to normal for the remainder of the treatment period.Although, the brief elevation in TGFβ-1 and CTGF expression in the earlier stages of our diabetic exposure most likely induced increased ECM production, it did not however explain continued up-regulation of cardiac ECM components despite the normalization of TGFβ-1 and CTGF.The results suggested that there were other mechanisms in place that post-translationally stabilize cardiac ECM proteins.These findings lead us to examine cardiac MMP and TIMP expression.We observed decreased MMPs and increased TIMPs suggesting ECM degradation was diminished in the embryonic hearts exposed to STZ-induced GDM, due to an increase in MMP inhibition by TIMPs.Thus, we propose the following model; the initiation of increased ECM expression appears to be caused by elevated glucose levels which induce TGFβ-1 and CTGF expression.Following the initiation of increased cardiac ECM production, protein stabilization of ECM components by MMPs and TIMPs occur in the embryonic heart.DM is a disease that has been proven to be detrimental to heart and kidney function in adults, in which associated fibrosis causes organ dysfunction.We have shown that GDM causes pre-fibrotic conditions in the developing heart which may affect septum and valve formation. In addition, fibrosis of the developing heart resulting from GDM may also influence the efficiency of organ function in these offspring.