Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

ROLE OF CAVEOLIN-3 IN THE MODULATION OF HERG POTASSIUM CHANNEL

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr) that is important for cardiac repolarization. Previously, we have discovered that hERG channels rapidly internalize in low extracellular K+ ([K+]o). In cell culture, this process is driven by the endocytic protein, caveolin-1 (Cav1), which is an integral player in the caveolae-dependant endocytosis pathway. However, in the heart, Caveolin-3 (Cav3) is, in fact, the predominant form in the myocyte, and thus may play a direct role in regulating hERG expression in the heart. Thus, I hypothesize that this reduction of hERG conductance in cardiac myocytes derives from the presence of Cav3, which is integral regulator of hERG homeostasis innately in the heart. To investigate the effect of Cav3 on hERG, I overexpressed Cav3 in human embryonic kidney 293 (HEK-293) cells stably expressing hERG channels. Cav3 overexpression significantly and specifically decreased both the hERG current amplitude and the mature channel expression in normal culture conditions. Co-immunoprecipitation analysis and confocal imaging demonstrated an association between hERG and Cav3 in HEK cells as well as rat and rabbit cardiomyocytes. Mechanistically, I discovered that Cav3 possesses a faster turnover rate compared to Cav1, and can enhance hERG degradation through up-regulating mature channel ubiquitination via the ubiquitin ligase, NEDD4-2. Knockdown of Cav3 in neonatal cardiac myocytes also enhanced hERG expression. My data indicate that Cav3 participates in hERG trafficking, and is an important regulator of hERG channel homeostasis in cardiac myocytes. This information provides a platform for future intervention of the hERG-induced type-2 long QT syndrome (LQTS).