Current Postdoctoral Fellows

Krista Blackwell, Ph.D.
Mentor: Paul McDermott, Ph.D., Department of Medicine, Division of Cardiology
Hypertrophic growth is dependent on selectively regulating the expression of proteins required for cardiocyte growth. The research is centered on the hypothesis that an increase in load enables the preferential translation of weak mRNAs, many of which encode for proteins required for cardiocyte growth. Translational mechanisms accelerate protein synthesis by increasing the activity and the amount of translational components such as ribosomes and initiation factors. My project will focus on examining the link between structural elements in the 5’-UTR and specific proteins involved in regulating translational efficiency by measuring the interactions between eIF-4E and mRNP particles. Efficient translation is dependent on mRNA: protein interactions that occur within the functionality of ribonucleoprotein particles. Immunoprecipitation, Western blotting and real-time RT-PCR methodologies will be the techniques used to carry out this study. Immunoprecipitation will allow the isolation of mRNP complexes using HA-4E/WT and HA-4E/K119A mutants. Real-time RT-PCR will identify class I (GAPDH) and class III (c-jun, c-myc) mRNAs associated with the complex in neonatal and adult cardiocytes.

Marisa Covington, Ph.D.
Mentor: Donald Menick, Ph.D., Department of Medicine, Division of Cardiology
Cardiac hypertrophy is an adaptive response to pressure or volume overload in the heart. However, heart failure occurs if the compensatory growth response fails to adequately normalize wall stress or the load is too great for hypertrophic growth to normalize. When this occurs a change in gene expression negatively impacts calcium handling, contractile function, and metabolism, specifically the Na⁺-Ca²⁺ exchanger (NCX1). Although exchanger is one of many gene expression changes that contribute to the eitology of heart failure, recent work has demonstrated the upregulation of NCX1 directly contributes to contractile disfunction. We have found that histone deacetylases (HDACs), specifically HDAC5, play a role in the regulation of NCX1 expression in the heart. The long range goal is to provide insight into the factors involved in regulating changes in gene expression contributing to heart failure. We propose to elucidate the molecular mechains by which HDAC5 mediates the upregulation of NCX1 expression in hypertrophically stimulated adult cardiocytes.

During the past several months, we have investigated if HDAC5 is required for NCX1 basal expression and/or NCX1 upregulation in response to hypertrophic agonists. We have constructed viral HDAC5 shRNA that decreases mRNA expression by 50% in adult cardiomyocytes. We are currently optimizing this technique and examining the effects of HDAC5 silencing on NCX1 expression levels, histone acetylation, and cardiocyte growth and cellular morphology. In addition, we have looked at the differences of HDAC inhibition with trichostatin A (TSA) in neonatal and adult cardiomyocytes. Treatment of neonatal cardiomyocytes with TSA resulted in a dose dependent inhibition of PE-stimulated upregulation of ANF, where as in adult cells TSA did not change PE-stimulated upregulation of ANF. In fact, over expression of HDAC5 resulted in a synergistic activation of ANF promoter activity in adult cardiomyocytes. In parallel, TSA inhibited phosphorylation of S6 in neonatal cells suggesting a decrease in protein synthesis, a measure of hypertrophy. In contrast, TSA increased phosphorylation of S6 in adult cardiomyocytes. We are currently investigating the differences of HDAC regulation of gene expression in neonatal and adult cardiomyocytes and the role they play in cardiac hypertrophy.

Andrew Hunter, Ph.D.
Mentor: Robert Gourdie, Ph.D., Department of Cell Biology and Anatomy
The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues, but little is known about the molecular mechanisms that control the size and distribution of gap junctions. I am addressing this issue by perturbing the interaction between connexin 43 (Cx43), the most abundant and well-studied connexin, and zonula occludins-1 (ZO-1), a MAGUK protein thought to link connexins to the actin cytoskeleton. To assay the role of Cx43-ZO-1 interaction in gap junction patterning, I am using connexin-deficient HeLa cells. HeLa cells expressing exogenously introduced wild-type Cx43 form small, discontinuous gap junctions that show peripheral co-localization of endogenously expressed ZO-1. By contrast, cells expressing Cx43-EGFP form large, sheet-like gap junctions that exclude ZO-1. However, the effect of Cx43-EGFP on both gap junction size and peripheral co-localization of ZO-1 is rescued by co-expression of untagged wild-type Cx43. Thus fusion of EGFP to the C-terminus of Cx43 alters gap junction size, possibly by masking the C-terminal amino acids of Cx43 that comprise the ZO-1 binding site. These results suggest that binding of ZO-1 to the C-terminus of Cx43 regulates the size and distribution of gap junctions, possibly by forming a dynamic mechanical link with the actin cytoskeleton, and/or by recruitment of signaling molecules to gap junctions. I am currently testing these ideas using mutant constructs and peptide inhibitors that directly target the interaction between Cx43 and ZO-1. Molecular interactions within the gap junctions of perturbed cells will be monitored using FRET microscopy, and live cell imaging will be used to follow the dynamic process of gap junction remodeling in real time.

Jason C. Mussell, Ph.D.
Mentor: W. Scott Argraves, Ph.D., Department of Cell Biology and Anatomy
I am working on a project, under the direction of Dr. W. Scott Argraves, seeking to determine the importance of cubilin in development, with an emphasis on cardiac and vascular development. Cubilin is a receptor involved in the metabolism of high density lipoprotein (HDL), intrinsic factor/B12, bone morphogenetic proteins, and a wealth of other molecules. Homozygous null mutants suffer embryonic lethality at ~8.5dpc. Our current hypothesis on cause of lethality is a misallocation of mesoderm that leads to defects in the endothelial (vascular) lineage. Mice heterozygous for the cubilin mutation suffer a broad spectrum of abnormalities including: pericardial effusion, cardiac defects, craniofacial defects, and neural tube disorders. Currently, I am describing the phenotypes of these animal models, as well as elucidating the biochemical pathway by which cubilin insufficiency produces abnormalities and/or lethality. As part of my coursework, I have attended, and presented at, the Cardiovascular Journal Club run by Dr. Paul McDermott.

Kevin Shores, Ph.D.
Mentor: Daniel R. Knapp, Ph.D., Department of Cell and Molecular Pharmacology
My initial project will be to apply a new differential expression proteomic analysis method (using iTRAQ reagents, "isotope tags for relative and absolute quantitation") to studies of cardiac hypertrophy in Dr. Zile's laboratory. We will apply this methodology to identify differentially expressed proteins in comparisons of normal ventricular muscle with muscle from volume overload and pressure overload models of hypertrophy in rats. Following extraction from cardiac muscle tissue, proteins will be digested and labeled for quantitative analysis with the iTRAQ reagents. Labeled peptides will be separated by strong cation exchange (SCX) chromatography on an Agilent 1100 HPLC system using a PolySulfoethyl A column. Fractions from the SCX column will be analyzed by liquid chromatography - mass spectrometry using an Agilent 1100 Series HPLC system interfaced via an electrospray ionization source to an ABI QSTAR mass spectrometer. The iTRAQ reagents contain two separate groups, a reporter group and a peptide reactive group. The reporter group contains isotopically labeled carbons and nitrogens resulting in its m/z ranging from 114 to 117. Each reporter group is balanced with a peptide reactive group such that the m/z of all iTRAQ reagents is 145. All variants of the iTRAQ reagents exhibit the same chromatographic characteristics, hence iTRAQ labeled peptides elute from the HPLC column simultaneously. Up to four cardiac muscle protein samples will be individually labeled and combined prior to chromatographic separation and MS analysis. The peptide reactive group chemically links to each lysine side chain and N-terminal group of a peptide, allowing for complete labeling of peptides in a sample. MS/MS analysis of individual peptides results in fragmentation of the iTRAQ reagent. A peak from the reporter group fragment appears in the mass spectrum between 114 to 117 m/z, while multiply fragmented peptide ions provide sequence information. Quantitative comparison of reporter group peak heights using ProQUANT Software (Applied Biosystems) provides protein expression ratios of up to four individual samples. Through use of this method, accurate comparison of protein expressions of the different cardiac muscle tissue samples will be performed. The resulting information will reveal the proteins for which expression is increased or decreased in the different hypertrophy models, one of which leads to compensated heart failure and the other to uncompensated heart failure. This information should contribute to the understanding of the mechanisms of heart failure and provide information that could be useful in the design of new therapies or preventive measures.

William James Tuxworth, Ph.D.
Mentor: Dhan Kuppuswamy, Ph.D., Department of Medicine, Division of Cardiology
The main theme of our research at Gazes Cardiac Research Institute is to gain a better understanding of the causes and consequences of load-induced cardiac hypertrophy. The onset of cardiac hypertrophy, as it manifest into pathological hypertrophy, is considered a promentant marker of heart failure, a disease that affects over 4.8 million Americans. In the laboratory of Dr. Dhan Kuppuswamy our primary goal is to determine how an increase in load is transduced by integrins to the modulation of intracellular signals that are responsible for the increase in mass associated with cardiac hypertrophy. As a post-doctoral fellow, I am studying the linkage between mechanical loading and increases in protein synthesis, an absolute requirement and hallmark of hypertrophy. Specifically, I am investigating the role that focal complex formation and subsequent S6K1 activation play in regulating increases in protein mass seen in hypertrophy. This sustained growth is thought to be achieved in part by enhancing the translation of specific 5'TOP (5' terminal oligopyrimidine tract) mRNA that encode for proteins important for ribosomal biogenesis. To this end, I have combined sucrose gradient fractionation with real-time RT-PCR to determine the distribution and mobilization of specific mRNA within a polysome profile. In a manuscript that is in preparation, I show that the translation of ribosomal protein L32 is dynamically regulated by pressure overload. Within the time frame of 24-48 hrs of pressure overload I see a significant increase in polysomal-bound L32 mRNA. Interestingly, these levels recede back to control levels starting at 1 week and attain levels similar to control at 4 weeks. When monitoring components involved in the regulation of L32 mRNA mobilization, I found that S6K1 activity parallels this pattern of mobilization. S6 activity, a target of S6K1, is higher than control levels at 24 and 48 hrs, but not to the extent as seen with S6K1. S6 protein expression increase 3-fold within 48 hrs, maintaining these new steady-state levels out to 4 weeks, indicative of increases in translational capacity required to maintain higher levels of protein synthesis to sustain mass.

In addition to this project, I am also involved in a study to determine if integrin activation and subsequent focal complex formation can contribute to hypertrophic growth via cellular redistribution of translational and ribosomal components. Positive results would support an intriguing mechanism in which cardiac myocytes can sense increases in load at the site of integrin engagement and subsequently direct components of the protein synthesis machinery to specific micro-compartments in a spatially restricted manner. Such translational events can eventually lead into the site-directed synthesis of sarcomeric proteins where a cell senses a change in load. Indeed, preliminary results using Western blot analysis show that the eukaryotic initiation factor 4E (eIF4E), a component of the 48S pre-initiation complex responsible for binding and transporting mRNA to the ribosome, undergoes a redistribution whereby more eIF4E is found in the cytoskeletal and membrane fractions during pressure overload. Studies are currently underway to confirm this result by using confocal microscopy.

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