Current Predoctoral Fellows
Anne Deschamps, B.S.
Mentor: Francis G. Spinale, M.D., Ph.D., Department of Surgery
Left ventricular (LV) remodeling is a common structural event following myocardial ischemia and reperfusion (IR), which can lead to congestive heart failure (CHF). Molecular and cellular mechanisms leading to LV remodeling are many. The induction of a number of neurohormonal pathways, loss of myocytes viability, and activation of matrix metalloproteinases (MMPs) all contribute to the remodeling process. MMPs are a family of proteolytic enzymes capable of degrading all components of the extracellular matrix (ECM). Both soluble and membrane-bound MMPs exist, with membrane-type 1-MMP (MT1-MMP) being prototypical. Because MT1-MMP has a broad substrate specificity, it can degrade many extracellular matrix (ECM) components including fibronectin and laminin. Notably, MT1-MMP can hydrolyze type I collagen into its characteristic 1⁄4 and 3⁄4 fragments contributing to ECM degradation.
MT1-MMP has been shown to be important in proteolytic activation cascades and can directly activate MMP-2 and –13 and indirectly MMP-9. Aside from ECM degradation, MT1-MMP is capable of acting on non-ECM proteins and signaling molecules such as tumor necrosis factor-α. While mice deficient in other MMP species show little phenotypic change, MT1-MMP deficient mice show an extremely disfigured phenotype due to inadequate collagen turnover and death by 3 weeks of age. This mouse model demonstrated the critical importance of this protease during the developmental process and raises the issue about the effects of increased MT1-MMP levels with pathological processes such as IR. The central hypothesis of this project is that the induction of MT1-MMP is temporally related to changes in oxygenation and that a specific portfolio of upstream mediators regulates these changes with IR.
My research will determine the activity of MT1-MMP in vivo in a porcine model of IR. We will also establish a validated model of real-time fluorescence (i.e. activity) detection by in vivo assay of porcine myocardium. Regulatory mechanisms regarding MT1-MMP activation using a myocardial fibroblast culture system will be studied. And finally, we will directly measure the activation pattern of MT1-MMP in vivo in patients undergoing cardioplegic arrest followed by reperfusion.
Loretta Hoover, B.S.
Mentor: Steven W. Kubalak, Ph.D., Department of Cell Biology and Anatomy
A significant proportion, 1:100 of newborn infants suffer from cardiac anomalies making it the most frequent type of birth defect. Abnormal valve and septa formation comprise the majority of these anomalies. Two knockout mouse models that we are using, transforming growth factor beta-2 (TGFβ2) and retinoid X receptor alpha (RXα), suffer from cardiovascular abnormalities including improper formation of the outflow tract (OFT) septum. Our lab has previously shown the RXRα-/- has increased levels of mRNA and protein for TGFβ2 in the embryonic heart at the time that OFT septation is occurring. These embryos also have elevated apoptosis in the developing endocardial cushions of the OFT. Using whole mouse embryo culture we have linked elevated TGFβ2 with enhanced apoptosis in the endocardial cushions of wild-type mice 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 and remodeling in the developing endocardial cushions of the OFT. Understanding how the TGFβ and retinoic acid (RA) signal pathways interact is a primary goal of this project.
To study activation of signaling cascades initiated by TGFβ2 and RA 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 TGFβ-/- 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 and Smad3 in isolated heart cells, and evaluating this effect in the presence and absence of RA.
Preliminary experiments have revealed a previously unknown role for RA in TGFβ signaling. Normally, effects of activation by RA are observed several hours after exposure. This time lag is due to obligatory transcriptional events that must be set in motion via RA receptors. However, when we simultaneously treat cells with TGFβ2 and RA for only one hour (too short for transcriptional events to contribute to a response), we observed a synergistic activation of Smads by TGFβ2. We are further exploring this response to TGFβ2/RA to determine the possible significance of this effect during cardiac development. 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.
Ira M. Mains, M.D.
Mentor: Francis G. Spinale, M.D., Ph.D., Department of Surgery, Division of Cardiothoracic Surgery Research
Myocardial remodeling, as defined by changes in the structure of the left ventricle (LV), is characterized by both structural and spatial alterations LV myocytes as well as structural and compositional changes in the extracellular matrix (ECM). The primary cell type responsible for modulating the ECM in myocardium is the myocardial fibroblast (MF). The specific contribution of the MF towards modulating myocardial ECM in either a normal or congestive heart failure (CHF) setting is unknown. MFs modulate ECM by synthesizing ECM components and matrix metalloproteinases (MMP), a class of proteases responsible for ECM degradation. Previous studies in human patients with CHF have demonstrated an increase in multiple MMP types as well as increased levels of serum markers of collagen degradation, indicative of enhanced ECM turnover. Expression of multiple MMP types and ECM components are modulated by several classes of extracellular signaling molecules including, neurohormones, cytokines, and growth factors; all of which are changes in patients with CHF. However, the potential for these molecules to modulate the ECM remodeling via MF has not been performed in human normal or CHF MFs. Using primary cultures of human MFs established from age and site matched LV of explanted hearts from patients with dilated cardiomyopathy or from normal patients (ejection fraction > 55%) undergoing elective coronary artery bypass surgery through LV myocardial biopsies of the normally perfused region of the anterior free wall, this study is examining ECM modulatory differences between normal and CHF derived MF after both steady state and exposure to bioactive molecules. Parameters of ECM synthesis and degradation are being measured at both the level of the protein and transcript. These studies will provide the necessary foundation for future investigation into the intracellular signaling pathways responsible for aberrant ECM modulation and will provide a cell-based platform for the discovery of novel therapeutic targets in CHF.
Phillip Moschella, B.S.
Mentor: Dhandapani Kuppuswamy, Ph.D., Department of Medicine, Division of Cardiology
This project will add to the knowledge surrounding the complex signaling cascade that is responsible for the most common cause of death in all industrialized nations, namely cardiac failure that stems from pathological hypertrophy. Understanding the complete pathway will lead to new pharmaceutical interventions and the possibility of better treatment and possibly a cure.
In response to an increased mechanical load on the heart, the terminally differentiated cardiac myocytes alter their pattern of gene expression to increase protein synthesis, and size. This compensatory expression initially normalizes wall stress, but becomes an independent risk factor for cardiovascular mortality and morbidity. In addition to mechanical stress it is accepted that a variety of chemical signals, like Endothelin-1 (Et-1), initiate signaling cascades that act concurrently in vivo and are independently responsible in cell culture models for the changes seen in cardiac hypertrophy.
Of major concern in cardiac hypertrophy is the increase in protein synthesis, which can occur via accelerated protein translation, and/or increased ribosomal biogenesis. This increase in ribosomal biogenesis is controlled by phosphorylation of the 40 S ribosomal S6 protein by S6K1 (otherwise referred to by its isoforms p70 S6 kinase (p70S6K) or p85 S6 kinase (p85S6K). At least eight multiple sequential phosphorylations are required for kinase activation on S6K1. Preliminary data has linked Protein Kinase C epsilon (PKC epsilon) through the classical ERK pathway to phosphorylation at the Ser/Thr-421/424 residues while phosphorylation occuring at the Thr-389 site is rapamycin (an mTOR antagonist/inhibitor) sensitive and possibly linked to PKC delta (unpublished data). Recently it has been discovered that there is a specific target of S6K1, namely SKAR that has profound effects on cell size.
Over the past year we have characterized the specific PKC isoform contributions to S6K1 activation. We have discovered the contributions of the novel PKC isoforms, PKC epsilon and PKC delta, on several specific phosphorylation sites on S6K1. A manuscript is now in preparation. We have also created two antibodies against SKAR, one N-terminal and one near the C-terminus. We have begun to characterize SKAR and its phosphorylation state in pressure overloaded myocardium using 2-D western analysis and confocal imaging. Concurrently we have also begun to develop several immunoprecipitation protocols using these same antibodies. Recently we have created some si-RNA constructs against S6K1 and we will begin to use these in some cell culture models of pressure overload, and look at the activation of SKAR and its effect on cell size. Also we have determined the nucleotide sequence for SKAR and will begin to make mutants of these phosphorylation sites and see if they have any effect on cell size and what effect mutating or truncating SKAR will have on its downstream effects.
J. Matthew Rhett, B.S.
Mentor: Robert Gourdie, Ph.D., Department of Cell Biology and Anatomy
Cardiac regeneration is simultaneously one of the most interesting and controversial areas in heart research today. Cardiac infarction usually results in an inflammatory and fibrotic response. Much like the case in epidermal wound healing the initial inflammatory response can cause a great deal of damage. This is evidenced by a decreased infarct size in neutrophil depleted animals undergoing reperfused myocardial infarction. Following the inflammatory response fibroblasts migrate into the infarct area and proliferate resulting in a fibrotic scar. Excessive scarring and collagen are major contributors to left ventricular dysfunction after myocardial infarction. Based on this knowledge it is reasonable then to speculate that reduction of inflammation and scarring could improve the prognosis for patients post myocardial infarction. In the Gourdie lab we have developed a novel peptide termed ACT-1 that targets basic cellular processes like migration and proliferation specifically in fibroblasts and inflammatory cell types. ACT-1 has shown considerable promise in accelerating healing and tissue regeneration following skin wound. My proposed study is to evaluate the potential beneficial effects of this peptide in myocardial repair following infarction.
Ongoing work in the Gourdie lab indicates that ACT-1 may alter the ability of fibroblasts to migrate and/or proliferate. Work with skin wounds in mice show an increased healing time in ACT-1 treated versus control wounds. We believe this may be due to decreased inflammation because of a decreased ability for neutrophils to extravasate based on similar work by Qui et al. Third, members of the Gourdie lab have initiated a study in which left ventricular cryogenic wounding followed by treatment with ACT-1 is performed on rats. Future work will include assays that are designed to separate out the effects of ACT-1 on proliferation and migration. Specifically, a proliferation assay and a transwell migration assay using primary cardiac fibroblast cells with or without ACT-1 treatment. Furthermore, collagen synthesis will be assayed in the same cardiac fibroblasts under similar treatment conditions to assess possible effects on scarring.
Elaine Wirrig, B.S.
Mentor: Arno Wessels, Ph.D., Department of Cell Biology and Anatomy
The atrioventricular (AV) junction is the cardiac segment situated between the atria and the ventricles in the developing heart. Almost 20% of all congenital heart abnormalities affect the tissues of the AV junction. This includes malformations of the AV valves and the AV septum, most of them involving the tissues derived from the AV endocardial cushions. The AV cushions initially develop by local subendocardial accumulation of extracellular matrix (ECM) at the AV junction. Subsequently, they become populated by cells as a result of epithelial-to-mesenchymal transformation (EMT). In this process a subset of cushion endocardial cells transforms, assumes a mesenchymal phenotype, and migrates into the ECM. The specific Aim of this project was to identify novel candidate genes involved in the development of atrioventricular cushion tissues. A DNA microarray, using RNA isolated from AV junctional and ventricular tissue of the mouse at ED11.0, has resulted in a set of 136 genes that may be important in the development of the AV cushions/junction.
We have recently initiated studies to characterize two of these genes, UGDH (UDP glucose dehydrogenase) and CRTL1 (Cartilage Link Protein 1). Our preliminary in situ hybridization studies indicate that UGDH is relatively abundant in the endocardium at ED9.5 and is also expressed in the cushion mesenchyme after EMT. As UGDH has been shown to be important for hyaluronan synthesis in other tissues and hyaluronan is a major component of the ECM of the cardiac cushions, our results indicate a hitherto unrevealed role for UGDH in mammalian heart development. CRTL1 has been previously described as being an important extracellular matrix (ECM) protein that stabilizes the interactions between proteoglycans and hyaluronan. Our immunohistochemical studies show for the first time that CRTL1 is expressed in AVC cushions as well as the cushions of the OFT. Given their expression pattern, we believe that CRTL1 and UGDH are important for development of the AV/OFT cushions. Future studies will be focused on determining their role in pre- and post-EMT cushion development, specifically focusing on their involvement in spatiotemporal regulation of the AV cushions, which will include mouse models in which these genes have been perturbed.
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