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Kenneth R. Boheler, Ph.D., Investigator Head, Molecular Cardiology and Stem Cell Unit Laboratory of Cardiovascular Science E-mail: bohelerk@mail.nih.gov |
| Biography: Dr. Boheler received his B.Sc. from Duke University and his Ph.D. in Physiology and Pharmacology from the University of California, San Diego. After completing a post-doctoral fellowship and working as a Researcher in Molecular Biology at Unit 127 of the National Institutes of Health and Medical Research (INSERM) in Paris, France, he was appointed Assistant Professor (Lecturer) at Imperial College School of Medicine in the Department of Cardiothoracic Surgery, London, United Kingdom. In October 1996, he joined the NIH in Baltimore to head the Molecular Cardiology Unit of the Cardiac Function Section of the Laboratory of Cardiovascular Science. |
| Overview: The main focus of this unit is to define the molecular bases of aging in the heart. Many features of the age-associated changes in heart cells resemble those found during fetal development. For this reason, emphasis has been placed both on studies of development and on that of aging. The focus on early cardiac gene expression has relied greatly on the use of an embryonic stem (ES) cell differentiation model system. In these studies, potential early cardiac gene transcription factors will be identified and the proteins responsible for activating expression are being targeted using standard molecular biological techniques. For aging, a number of model systems are being developed so that specific genes can be targeted during senescence to examine their functional consequences. Each project area has multiple components, and it is hoped that through integration of developmental with aging studies, we will be able to obtain a global view of cardiac gene expression and how alterations in individual gene expression patterns lead to physiological and pathophysiological consequences. |
Manuscripts: Anisimov, S.V., Tarasov, K.V., Tweedie, D., Stern, M.D., Wobus, A.M., Boheler, K.R.: SAGE Identification of Gene Transcripts with Profiles Unique to Pluripotent Mouse R1 Embryonic Stem Cells. Genomics 79(2): 169-176, 2002. -- Supplements A, B, and C (pdf) Sheydina A, Volkova M, Jiang L, Juhasz O, Zhang J, Tae HJ, Perino MG, Wang M, Zhu Y, Lakatta EG, Boheler KR. Linkage of Cardiac Gene Expression Profiles and |
| The focus of our research program over the past several years has involved examination of the expression and regulation of a number of proteins involved in regulating calcium movements in cardiac myocytes, including the sarcoplasmic reticulum calcium ATPase (SERCA), the Na/Ca exchanger (NCX1) and the sarcoplasmic reticulum calcium release channel (Ryanodine Receptor). The work has involved examination of the spatial and temporal expression of these mRNAs and proteins in the developing myocardium. Using simpler in vitro models, the regulation of presence of the mRNAs encoding some of these gene products has been studied through examination of signal transduction pathways. |
| Our work is focused on use of an in vitro differentiation model of mouse embryonic stem cells and embryonic carcinoma cells in an attempt to further understand the consequences of development and of altered gene expression on function of these proteins. Additionally, research in the laboratory has led to the development of mouse models having temporal and spatial control of gene expression. This system is currently being tested and plans are underway to actively apply this system to mouse transgenic models and to differentiating ES cells. Our recent experiments have exploited functional genomics to examine differentiation, development, aging and disease. |
| Signal Transduction Pathways Mediating SERCA2 Expression: We have exploited the human SERCA2 promoter to examine this gene's regulation in vitro. Transfection into neonatal rat cardiomyoctyes of the 2.8 kb human SERCA2 promoter constructs linked to reporter sequences indicate a lack of response with any of the adrenergic agonists, but regulation via sp1 and sp3 transcription factors has now been shown. Recent studies with Nuclear run-on assays have also indicated that transcriptional control of SERCA2 gene expression is not the primary mechanism responsible for increased mRNA, protein and function of SERCA2 seen perinatally. Studies are underway, to elucidate the mechanisms responsible for the post-transcriptional regulation, one possibility of which may relate to an alternatively spliced isoform of SERCA2 seen in the fetal myocardium, whose expression is greatly reduced late in gestation. |
| Expressional Analysis of Cardiac NCX in Development and Senescence: We have examined the mRNA expression of the Na/Ca exchanger (NCX) in rat heart during perinatal development and with aging. NCX is highly expressed in late fetal and neonatal rat hearts, decreasing to adult levels by 20 days after birth. The lowest level of accumulation is seen in 6 and 18 month old animals. In the 24 month old senescent rat, NCX expression is increased by almost 50% above that seen at 6 and 18 months (p<0.05), but is not different from that at 15 neonatal days. Results from nuclear run-on assays indicate that NCX expression during the perinatal period is regulated at least partially through transcriptional mechanisms. Relatively high transcriptional activity is seen at birth but by 20 post-natal days, no transcriptional activity from NCX can be detected. During development, there are no major changes seen in the use of the five identified transcription start sites, nor is there any major difference in the splicing patterns seen in the 5' untranslated regions. We have identified the presence of five different splicing variants in the cytosolic loop of the coding region, three of which have not been previously described in heart. We have also recently cloned a 2.8 kb fragment containing the putative cardiac NCX1 promoter and a consensus thyroid hormone responsive element which we are now examining. The work is now focused on the in vitro examination of this promoter. A number of putative GATA binding sites and Nkx binding sites have been identified. In transfection studies, GATA 4, 5, and 6 isoforms have been shown to be sufficient to transactivate this sequence. Constructs lacking these cis-binding elements or mutants of these sequences have been prepared and are being examined both in vitro and in the transgenic models described above. |
| Embryonic Stem Cells and Myocardial Development: This research area involves a model of in vitro differentiation of cardiomyocytes originating from embryonic stem cells (R1) and embryonic carcinoma cells (P19). The research is aimed at understanding the developmental processes involved in cardiac myocyte differentiation and development. To identify, atrial versus ventricular like cells, expression vector constructs have been made that link atrial and ventricular markers to the green fluorescence protein (GFP) and other selection markers. These constructs have been introduced into ES cells and positive transformants identified through neomycin resistance selection. From this work, we hope to use various molecular techniques to identify and analyze various transcription factors and growth factors that promote cardiac cell division and differentiation and importantly, the sequence of their activation and inhibition. Specifically, we are examining the expressed sequences of differentiating P19 cells through a technique called serial analysis of gene expression (SAGE). This technique takes advantage of PCR and type II restriction enzymes to isolate short sequences sufficient to identify RNA products expressed at any time point. Currently, SAGE analyses have been performed on adult mouse myocardium, 3+3 day in vitro differentiating P19 cells and a comparative analysis is underway with 3+0.5 day in vitro differentiating P19 cells. Through this technique, we hope to use the information gained about the expressed sequence pattern to target and specifically identify gene products that are important to cardiac differentiation. Using targeted ES cells, we have been able to demonstrate a role of the ryanodine receptor in the control of heart rate development. |
| Temporal/Spatial Regulation: The aim of this program is to develop conditional and inducible gene targeting models, limited to specific cardiac lineages (e.g. ventricular myocytes) and inducible at a desired developmental stage. The tools chosen to accomplish this program are the Cre Recombinase-Lox P recombination system and the tetracycline trans-activator system. A number of mice have been prepared that carry the Cre recombinase transgene under control of a tetracycline-sensitive promoter. Secondly, a targeting construct containing LoxP sites has been prepared such that induction of Cre Recombinase expression by withdrawal of tetracycline should cause excision of a critical exon in a targeted gene. This system has been placed under control of a lineage-specific promoter so that a tissue-specific knockout can be made to occur at a specified time. Currently a tetop-Cre Recombinase and MLC2V-tTA construct has been injected into pronuclei of C57BL/6 oocytes and a number of founder lines positive for these transgenes have been identified. These lines are currently being studied for appropriate expression using another reporter mice. To inducibly knockout RyR2 expression, a 15 kb mouse 129/SvJ genomic DNA fragment has been cloned, sequenced and the genomic structure determined. This sequence has been appropriately modified and lox P sites and neomycin resistance cassettes placed appropriately within the sequence. This mutant mouse RyR2 targeting vector has also been successfully introduced into embryonic stem cells, injected into blastocysts, and positive chimeras have been identified. This work is on-going. |
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