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.
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).
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).
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