Keywords

Gene expression, Heart -- Growth

Abstract

The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are essential for cardiovascular development as embryos lacking NE/EPI begin to die abruptly between embryonic days 10.5 and 11.5 due to apparent cardiac failure. The objective of this research aims to elucidate the mechanism of the embryonic fatality observed in the NE/EPI deficient mouse model. Here we utilized the dopamine β- hydroxylase knockout (Dbh-/-) mouse model, which lacks the gene and subsequent enzyme necessary for the conversion of dopamine to norepinephrine. Embryonic mouse hearts were extracted from Dbh+/+ (control) and Dbh-/- (experimental model) mice for mRNA transcript expression profiling. These studies were performed using the Affymetrix Mouse Genome 430A 2.0 Arrays and quantitative real-time RT-PCR. Gene expression data suggest a novel connection between the ability of the heart to synthesize adrenergic hormones and the gene expression of enzymes involved in the synthesis of retinoic acid. Specifically, we found a statistically significant change in transcriptional expression of the retinol binding protein-1 (Rbp-1), retinol dehydrogenase 12 (Rdh-12) and beta carotene monooxygenase-1 (Bcmo-1) genes in the E10.5 Dbh-/- mouse heart. The gene expression of Rbp-1 and Rdh-12 were increased 1.4 fold and 2.1 fold on the microarray, respectively. The proteins translated from these genes play central roles in the transport and enzymatic conversion of precursor molecules in the pathway of retinoic acid biosynthesis. Additionally, we found that the expression of Bcmo-1, an enzyme responsible for the breakdown of beta carotene to the retinoic acid iii precursor retinal, was down regulated 2.7 fold in the Dbh-/- heart based on microarray assessment. Bcmo-1 is a well known retinoic acid responsive gene, suggesting that the loss of adrenergic hormones in the Dbh-/- mouse heart may result in a deregulation of retinoic acid synthesis and further an alteration in the concentration of retinoic acid present in the embryonic tissue of adrenergic hormone deficient embryos. In addition, we utilized a genetic mouse model that expresses β-galactosidase (β-Gal) in cells capable of synthesizing epinephrine in order to identify the spatial and temporal distribution of adrenergic-derived cells in the developing heart. The model was designed so that cells capable of expressing the gene phenylethanolamine Nmethyltransferase (Pnmt), which is responsible for the synthesis of epinephrine, also produce the enzyme β-Gal as a reporter. The resulting presence of the β-Gal enzyme can be visualized using a β-Gal substrate called XGAL, which is converted into a blue precipitate when cleaved by the β-Gal enzyme. Evaluation of the location of these cells in the embryonic heart showed a preferential distribution at the atrioventricular sulcus at E10.5, and later at E18.5 a more widely distributed ventricular pattern was observed. In addition, the right atrium showed a cluster of XGAL positive cells (blue cells) near the region of the sinoatrial node, while the distribution of XGAL positive cells in the left atrium was quite diffuse. Interestingly, when the adult heart was examined, it was discovered that cells capable of synthesizing epinephrine (adrenergic-derived) are found predominately on the left side of the heart. This left-sided distribution appears to be non-random and non-uniform, since specific regions are consistently XGAL positive, but not every cell in each region. Whole mount and 3-dimensional reconstruction of the iv β-Gal staining showed that these cells traverse the depth of the heart at the midventricular and apical regions. This finding is quite interesting and may provide new knowledge about the functional and structural characteristics of the adult heart. One observation is that these cells may contribute to the cardiomyopathy known as TakoTsubo or "Broken Heart" syndrome. The syndrome is characterized by left ventricular dysfunction during bouts of stress. Also, of particular intrigue is the anatomical correlation of the adrenergic derived cells and the helical ventricular myocardial band (HVMB). Careful examination of the spatial and directional pattern of these cells within the myocardium suggests they contribute primarily to a specific section of the HVMB. The significance of this finding is yet to be uncovered. Taken collectively, this study has shown a novel connection between two crucial developmental signaling pathways. Adrenergic hormone and retinoic acid signaling can now be viewed as cooperative partners in the development of the embryonic heart. In addition, this study has also shown that adrenergic derived cells in the adult heart have a distinctive left-sided distribution, which is non-random, non-uniform, and shows interesting features suggesting an anatomical connection to the HVMB and a clinical association to Tako-Tsubo syndrome. These findings will appreciably contribute to the knowledge base of the scientific community

Notes

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Graduation Date

2011

Semester

Summer

Advisor

Ebert, Steven

Degree

Doctor of Philosophy (Ph.D.)

College

College of Graduate Studies

Department

Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0003987

URL

http://purl.fcla.edu/fcla/etd/CFE0003987

Language

English

Release Date

July 2016

Length of Campus-only Access

5 years

Access Status

Doctoral Dissertation (Open Access)

Subjects

Dissertations, Academic -- Graduate Studies, Graduate Studies -- Dissertations, Academic

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