Identifying the molecular basis for the thickened heart walls observed in Drosophila bearing hypertrophic cardiomyopathy mutations




Marisa Mercadante

Introduction

Infantile hypertrophic cardiomyopathy (HCM) is a disorder characterized by thickened but undilated left ventricle of the heart. In humans, certain severe cases of HCM have been linked to two different point mutations in the ELAC2 gene, named HCM1 and HCM2 (Haack et al. 2013). ELAC2 has been highly conserved throughout evolution. Drosophila melanogaster possess a homologous gene to ELAC2 called RNaseZ (Xie and Dubrovsky 2015). The normal amino acids which are altered in HCM1 and HCM2 are conserved in the Drosophila RNaseZ protein (Figure 1).

The Drosophila heart is a long contractile tube made up of two rows of cells (Rotstein and Paululat 2016). Heart cells stop dividing after embryogenesis. The heart undergoes massive restructuring and growth during the larval stages, but the number of cells remains constant (Rotstein and Paululat 2016).

Previous research in the Dubrovsky lab generated stocks of flies with each of the two HCM-linked mutations. Histological analysis of 3rd instar larvae showed that both HCM1 and HCM2 mutants had significantly thicker heart walls than wild type larvae. It remains unknown the method by which HCM mutant hearts increase in thickness.

The purpose of this study is to determine whether the thickness observed in hearts of HCM mutants is due to an increase in heart cell size or number of heart cells. Though normal Drosophila heart cells are non-dividing, it may be that the HCM mutation results in the activation of division in this tissue. This project studies seeks to answer this question by analyzing the expression of Cyclin E encoding transcript, a marker of cell division.


Materials and Methods
Third instar fly larvae bearing wild type and HCM genotypes were collected, and their hearts were dissected and stored in ethanol at -80°C. 13 HCM1 mutant hearts and 10 wild type hearts were collected in total. As a positive control, 5 HCM2 and 5 wild type 3rd instar larvae were collected.
RNA was extracted from samples using the Nucleospin® RNA II kit

RT-PCR was performed with primer sets designed to the housekeeping gene Rp49, the heart tissue marker gene Hand, and the cell division marker gene Cyclin E using QIAGEN® One-Step RT-PCR Kit.
RT-PCR products were visualized on 1% agarose gels, purified, and sequenced.

To determine if RT-PCR products of unknown origin were the result DNA contaminants, RT-PCR was performed with the reverse transcriptase (RT) enzyme destroyed by incubating the reaction mix at 95°C for 15 minutes prior to the reaction.

Results
Hand and CycE were expressed in wild type (WT) and mutant hearts as well as larval samples (Figure 2). There was no visible difference in expression of CycE between wild type and mutant hearts.

Rp49 mRNA product was present in all samples (Figure 3A). A larger product of 349bp was detected in heart tissue only (Figure 3A). DNA sequencing of this product revealed the presence of intron 2 sequence. To determine whether this product resulted from DNA contamination or was an alternative mRNA transcript, RT-PCR was repeated with the RT destroyed. This prevented RNA from being amplified and producing a product. After this reaction, the 287bp RNA product was absent from the gel, but the 349bp product was still visible (Figure 3B).

Discussion
The purpose of this study was to determine whether the thickness of heart walls observed in Drosophila with HCM-linked mutations is due to an increase in heart cell size or number. The project was unable to detect a difference in expression of the cell division marker gene Cyclin E in mutant hearts as compared to wild type. Wild type larval hearts are terminally differentiated, non-dividing tissue that grows solely through cell size increase. However, results show that Cyclin E was expressed in the wild type heart, indicating that this gene is not a good marker of cell division for study in the Drosophila heart.

RT-PCR results detected expression of the Hand gene in the analyzed tissue, confirming that larval dissection of hearts was successful. Expression of Hand mRNA in the whole larvae tissue reflects the presence of heart in these samples. Rp49 was also present in all samples, but a 349bp product that contained intronic sequence was present only in the heart samples. When RT-PCR was performed with the reverse transcriptase destroyed to prevent RNA amplification, this 349bp product was still present, thus concluding that it is the result of DNA contamination. The absence of the 287bp Rp49 RNA product in this reaction confirms that the reverse transcriptase destruction was successful.

At this time, I cannot draw conclusions as to whether the HCM mutant hearts are undergoing cell division. This question necessitates further study with more informative marker genes, as well as qRT-PCR analysis to quantify differences between wild type and mutant expression. Potential future markers could include Bub1, Polo, and Mad2, which express proteins that are vital to microtubule spindle function during cell division (Logarinho et al. 2004; Whitfield et al. 2006).


References
Haack, T.B., Kopajtich, R., Freisinger, P., Wieland, T., Rorbach, J., et al. (2013). ELAC2 Mutations Cause a Mitochondrial RNA Processing Defect Associated with Hypertrophic Cardiomyopathy. The American Journal of Human Genetics 93, 211-223.

Han, Z., Yi, P., Li, X., and Olson, E. N. (2006). Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis. Development and Disease 133, 1175-1182

Logarinho, E., Bousbaa, H., Dias, J.M., Lopes, C., Amorim, I., Antunes-Martins, A., and Sunkel, C.E. (2004). Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells. Journal of Cell Science 117, 1757-1771

Rotstein, B. and Paululat, A. (2016). On the Morphology of the Drosophila Heart. Journal of
Cardiovascular Development and Disease 3, 15.

Whitfield, M.L., George, L.K., Grant, G.D., and Perou, C.M. (2006). Common markers of
proliferation. Nature Reviews Cancer. 6, 99–106

Yu, L., Daniels, J.P., Glaser, A.E., and Wolf, M.J. (2013). Raf-mediated cardiac hypertrophy in
adult Drosophila. Disease Models & Mechanisms 6, 964-976

Xie, X. and Dubrovsky, E.B. (2015). Knockout of Drosophila RNase ZL impairs mitochondrial
transcript processing, respiration and cell cycle progression. Nucleic Acids Research 43, 10364-10375.

Figures


Figure 1-Figure 1 - Alignment of ELAC2 and RNaseZ demonstrating homology


Figure 2-Figure 2 - RT-PCR products of wild type (WT) and mutant heart and whole larvae RNA with CycE (A) and Hand (B) primers


Figure 3-Figure 3 - A) RT-PCR products with Rp49 primers. B) Products of RT-PCR of WT and mutant heart samples with Rp49 primers and RT destroyed by heat prior to the reaction.


Mutations in the human gene ELAC2 have been linked to hypertrophic cardiomyopathy (HCM), a deadly disease that causes thickened heart walls. Drosophila melanogaster possess a homolog to ELAC2 called RNaseZ. Previous research has demonstrated that HCM-linked mutations in fruit fly larvae also result in heart walls that are significantly thicker than observed in wild type. This study seeks to identify whether hearts isolated from flies carrying HCM mutations are thickened as a result of increases in cell size or cell division. Heart cells of wild type larvae are non-dividing, and as such should not express genes associated with cell division. Wild type and HCM mutant larval heart samples were tested for the presence of the Cyclin E transcript, a marker of cell division in Drosophila. Cyclin E transcript was expressed in wild type and mutant hearts, indicating that the expression of this gene is not a good marker for cell division in fly hearts. There was no detectable difference in expression of Cyclin E transcript in the different heart tissues.

Full Paper

Acknowledgments

I would like to thank Dr. Dubrovsky and Ekaterina Migunova for all their guidance in developing this project and dissecting, and for providing fly stocks. I would also like to thank Dr. Rubin for his guidance throughout this project. Lastly, I want to thank Anthony Evans and Devin Rocks for their constant help and support.


This document was last modified 05/14/2019.
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