Past Predoctoral Fellows

Jason Guichard, B.S.
Mentor: Craig Beeson, Ph.D., Department of Pharmaceutical Sciences
Diabetes is the fifth leading cause of death by disease in the U.S. and people with diabetes are at a higher risk for heart disease, blindness, kidney failure, and other chronic conditions. One of the initial pathological events during the onset of type II diabetes and related metabolic syndromes is insulin resistance and impaired glucose utilization in muscle. We have found that physiologically relevant concentrations of high density lipoprotein (HDL) enhance glucose oxidation and coupling between mitochondrial respiration and glycolysis in both skeletal and cardiac muscle. The coupling is manifested by increased lactate production upon mitochondrial respiratory inhibition (the Pasteur effect).

We hypothesize that HDL affects the expression and activation state of proteins involved in a supramolecular complex at or near the mitochondria that senses respiratory flux and couples this to glycolysis. Glucose oxidation, respiration, and oxygen consumption for rat myoblasts and adult feline cardiocytes were increased in response to treatment with HDL. HDL increased the coupling between respiration and glycolysis as measured by the increase in lactate production in response to blocked respiration in rat heart myoblasts and feline adult cardiocytes. Skeletal and cardiac muscle cells were treated with mitochondrial respiration inhibitors, gently lysed, and intact mitochondria were isolated. Supramolecular complexes obtained from isolated mitochondria were separated by size exclusion chromatography, resolved by SDS-PAGE, and several differential proteins correlated with HDL-induced coupling were identified. HDL clearly changes the phosphorylation state of the cell and mitochondrial isolates from mitochondrial respiration inhibited cells show differences between control isolates. The underlying biochemical mechanisms that regulate mitochondrial respiration and its coupling to glycolysis in muscle play important roles in the pathology and treatment of type II diabetes. Uncovering the identity of proteins and/or their binding partners will provide insight into HDL action and its contribution to the coupling between mitochondrial respiration and glycolysis, which will enhance our understanding of diabetes disease pathology and treatment.

Shantae Jenkins, B.S.
Mentor: Steven Kubalak, Ph.D., Department of Cell Biology and Anatomy
The retinoid x receptor alpha knockout (RXRα^-/-) mouse has been previously described as a model of congenital heart disease. The myocardium forms improperly in the RXRα^-/- and there is lack of septation of the outflow tract. The mutation results in embryonic lethality at midgestation (E13.5-15.5) from overall heart failure. Reactivation of RXRα in cardiomyocytes of knockout animals does not result in rescue of the thin myocardial phenotype. Recently, studies that inactivate RXRα mediated signaling within the epicardial cell lineage closely recapitulate the phenotype of the RXRα^-/-. Heretofore, it has not been demonstrated whether the epicardium of the RXRα^-/- develops properly. Thus, we have shown that the progenitor population of the epicardium, the proepicardium, is compromised in the RXRα^-/- prior to migration. Further, there is a delay in the epicardial covering of the heart in RXRα^-/-. After the epicardium has matured, there is an abnormal detachment from the underlying myocardium. Investigation of epicardial-specific markers and extracellular matrix proteins demonstrates that fibronectin is elevated in the developing proepicardium and subepicardial spaces of the RXRα^-/-. Recently, we have examined fibronectin expression in vitro in RXRα^-/- and wild type explants. Indeed, there is an altered localization of fibronectin in the mutant explant that is quite reminiscent of in vivo mutant proepicardial expression. Given this change, we performed studies to determine if altering the concentration of fibronectin in the environment of the proepicardium would result in delayed outgrowth of proepicardial-derived cells. These studies did not result in altered outgrowth suggesting that although fibronectin is altered in the mutant it does not prevent the initial outgrowth of the proepicardium. We have demonstrated that TGFβ2 regulates fibronectin expression within the developing proepicardium at the level of mRNA and protein. Taken together, our studies suggest that the increased fibronectin synthesis seen in the RXRα^-/- may in part be attributable to TGFβ2 but the elevation of fibronectin within the proepicardium is not responsible for the abnormal epicardial phenotype.

Christiana Kappler, B.S.
Mentor: Donald Menick, Ph.D., Department of Medicine, Division of Cardiology
Cardiac hypertrophy occurs when an excessive hemodynamic workload is placed on the heart. Although this is initially an adaptive response that allows the heart to maintain efficient function in the face of increased mechanical stress, it will ultimately deteriorate into cardiomyopathy and heart failure. Because cardiac myocytes become terminally differentiated post-natally, an increase in heart size must be achieved by an increase in size of each individual cell. In addition to an increase in cell size, there is a marked change in cardiac gene expression. In many models of heart disease and failure, the activity and protein level of the sodium calcium exchanger (NCX) is increased. While it has been suggested that this increase is initially a compensatory response that allows the cell to maintain calcium homeostasis, this adaptation has deleterious consequences. The primary hypothesis of my research is that alteration of exchanger activity and expression during cardiac hypertrophy is associated with molecular signal transduction pathways. Strikingly, recent findings in our laboratory have revealed not only that signaling factors have an effect on NCX expression, but that inhibition of reverse mode NCX activity by treatment with KB-R7943 (KBR) can modulate signal transduction pathways. There is still a great deal more to be understood, but clearly changes in exchanger expression are important. Therefore, we want to identify the molecular pathways that are associated with up-regulation of the exchanger gene in cardiac hypertrophy and failure.

Jessica Paulk Peterson, B.S.
Mentor: Steven Kubalak, Ph.D., Department of Cell Biology and Anatomy
One percent of newborn infants suffer from cardiac anomalies making it the most frequent type of birth defect. The majority of these anomalies result from abnormal valve and septum formation. We have two knockout mouse models, Transforming Growth Factor Beta2 (TGFβ2) and Retinoid X Receptor alpha (RXRα), that suffer from cardiovascular abnormalities including improper formation of the outflow tract (OFT) septum. We have previously shown RXRα^-/- has increased levels of TGFβ2 mRNA expression and protein in the embryonic heart at the time that OFT septation is occurring, and that these embryos have elevated apoptosis in the developing endocardial cushions of the OFT. Using whole mouse embryo culture it can also be shown that exogenously administered TGFβ2 in wild-type mice results in enhanced apoptosis in the endocardial cushions with a pattern analogous to the RXRα^-/-. Thus, we have a mouse model where TGFβ2 is downregulated and another model where TGFβ2 is upregulated, and can use these to determine the mechanism by which TGFβ2 regulates apoptosis in the developing endocardial cushions. Identifying which signaling pathway TGFβ2 utilizes to induce apoptosis will be important for determining key players of apoptosis in development, and also for determining which cells initiate apoptosis in the developing heart. These studies will also shed more light on the overall role for TGFβ2 during remodeling of the heart. To study activation of signaling cascades initiated by TGFβ2 we are characterizing midgestation trypsinized hearts, an in vitro model in which TGFβ2 also induces apoptosis. The use of this in vitro model as well as mouse embryonic fibroblasts derived from wild type, RXRα^-/-, and TGFβ2^-/- will help us to determine how TGFβ2 signals apoptosis in vivo and the role for this growth factor in the malformations observed in the mutants. We are currently investigating the ability of TGFβ2 to induce activation of Smad2, Smad3, and the mitogen activated protein kinases (MAPKs), which are known downstream transcriptional regulators of TGFβ2 signaling. Results from our studies will give insight into how normal septation of the OFT occurs, as well as how septation defects in congenital heart diseases arise.

Laura Spruill, B.S.
Mentor: Paul J. McDermott, Ph.D., Department of Medicine, Division of Cardiology
During hypertrophic growth of cardiac muscle, mRNAs are recruited into polyribosome complexes by mechanisms that increase translational initiation. A subset of mRNAs possesses an excessive amount of secondary structure in their 5´-untranslated regions that lowers their translational efficiency. Our hypothesis is that the mRNAs in this subset, which encode for proteins required for growth, must be selectively translated following a growth stimulus. My current work is an extension of the work completed last year using the transverse aortic constriction model of pressure overload cardiac hypertrophy, in which we identified 6 mRNAs that appear to be translationally regulated in response to this growth stimulus. We are currently focused on the translational regulation of c-jun, one of the mRNAs identified in the TAC model. We have been using a feline cardiocyte cell culture model to infect adenoviruses containing constructs encoding components of the translational machinery to manipulate the translational regulation of c-jun. These components include eIF4E, Mnk and a DN-Mnk, which are important for translation initiation. We are also in the process of creating synthetic adenoviral reporter constructs, containing the 5´ Untranslated Regions of ODC and c-jun upstream of the beta-galactosidase gene. The expression of these constructs in our feline cardiocyte model will allow us to specifically address the effect of the 5´UTR in the regulation of translation of these mRNAs during growth.

Sarah Sprunger, B.S.
Mentor: James A. Cook, Ph.D., Department of Physiology and Neuroscience
Research in our laboratory focuses on the role of altered innate immune mechanisms in the pathogenesis of septic shock. Ms. Sprunger's research specifically centers around the potential involvement of the phosphoinositide 3-kinase (PI3K) signaling pathway in the inflammatory response in septic shock. The PI3K pathway, including Akt, which is downstream of PI3K, may be involved in endotoxin tolerance, a phenomenon of decreased inflammatory response that promotes survival in the face of septic injury. Her research currently employs in vitro primary cultures of macrophages and cell lines, in vivo animal models of shock and transgenic murine models, along with pharmacological inhibitors of PI3K. These studies are designed to elucidate the importance of the PI3K pathway in endotoxin tolerance.

Ms. Sprunger's course work at MUSC has included Foundations of Biomedical Sciences and Essential Scientific Practices in a common first year core curriculum that provided instruction beginning at the cellular, tissue, and organismal level along with current techniques, laboratory practices, and scientific ethical considerations. Other courses have included Statistics, Technology Updates, Principles of Pharmacology, Integrative Biology of the Cardiovascular System and a seminar series entitled Important Unanswered Questions in the Biomedical Sciences. Currently she is enrolled in Dental Pharmacology and Advanced Topics in Cell Signaling.

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