26 Loss of FBXO44 Causes Cardiac Dysfunction

Faculty Mentor: Sihem Boudina (Nutrition and Integrative Physiology, University of Utah)

Abstract

Fbxo44 is a member of the F-box family which is involved with the specific degradation of certain proteins including the Regulator of G protein Signaling 2 (RGS2) in non-cardiac cells. Low levels of RGS2 are associated with hypertension and heart failure (Sjörgen et al., 2015). However, the role of Fbxo44 in the heart is currently unknown. Previous studies in the laboratory have shown that the deletion of PR domain-containing 16 (Prdm16) has resulted in a decrease in Fbxo44 expression in the heart which was associated with increased repetitive element expression and DNA damage. Further, recent studies in cancer cell lines have demonstrated that loss of Fbxo44 leads to de-repression of repetitive elements, resulting in increased DNA damage, activation of the interferon response, and ultimately, cell death. Thus, the goal for this research project is to test the overall hypothesis that Fbxo44 degradation in cardiomyocytes results in cardiomyopathy. We tested this hypothesis in two interrelated, yet independent experiments using in vivo and in vitro approaches.

Aim 1: the in vitro approach will consist of using Neonatal rat ventricular myocytes (NRVMs) isolated from male and female newborn rats, with gain and loss of function of FBXO44. To do so we will test the hypothesis that the deletion of FBXO44 in NRVMs will result in cardiac hypertrophy. We will use siRNA mediated gene silencing to target FBXO44 and adenovirus-mediated overexpression to increase FBXO44 levels. There will also be an siRNA with a sequence that does not recognize any gene (scrambled) and an empty vector which are used as control groups. We will measure cell size and expression of hypertrophic genes. These indices will be assessed as baseline, as well as after exposing the cells to a hypertrophic stimulus.

Aim 2: for the in vivo approach, we will use mice with conditional deletion of FBXO44 in cardiac cells. To do so we will test the hypothesis that the deletion of FBXO44 results in cardiac hypertrophy. Two genotypes will be used: non-Cre wild-type mice and αMHC-Cre FBXO44 knockout (KO) mice. We will measure the internal diameter, posterior wall thickness, and cardiac contractility by echocardiography. Quantitative real time PCR (qPCR) will be used to measure the mRNA expression of stretch markers in the heart which will help us determine whether the heart has experienced cardiac hypertrophy. Also, we will stain the lipid membrane with wheat germ agglutinin (WGA) to measure the cross-sectional area of cardiac cells. These experiments will be performed in both male and female wild type and KO mice.

Results

Echocardiography in mice at 16 weeks showed that loss of Fbxo44 caused cardiac dysfunction. Ejection fraction and fractional shortening were significantly decreased in Fbxo44 KO male mice when compared to age-matched wildtype controls. Both females and males showed a significant difference in Left Ventricle (LV) Internal Diameter during Diastole (LVID-d) with the Fbxo44 KO having an increase in this parameter suggesting an enlargement of the LV. Similar results occurred during systole, although the difference did not have significance in males.

Once NRVMs were isolated, we treated the cardiomyocytes with different concentrations of Fbxo44 siRNA. We found that 30 nM Fbxo44 siRNA effectively knocked down (KD) Fbxo44 in the NRVMs 72.5%. To test the specificity of our KD we measured the mRNA expression of another Fbxo gene, Fbxo40, and the results established that Fbxo40 has not been deleted in the cardiomyocytes whereas Fbxo44 was deleted. We ran a qPCR for stretch markers including Natriuretic Peptide A (Nppa) and Natriuretic Peptide B (Nppb). We found an upregulation of both Nppa and Nppb in Fbxo44 KD cells. We also found an upregulation in Myosin Heavy Chain 7 (Myh7), and Cyclin-dependent kinase inhibitor 1A (Cdkn1a) in the Fbxo44 KD cardiomyocytes, suggesting that these cells revert to a more fetal state. Lastly, we examined the mRNA expression of repetitive elements including Long Interspersed Nuclear Elements-2 (L1-2) and Long Interspersed Nuclear Elements-3 (L1-3) and found it rather decreased in Fbxo44 KD cells.

We performed western blot on the NRVM samples to detect specific proteins including Fbxo44 and Stimulator of Interferon Genes (STING). Fbxo44 KD cells had a significant decrease in Fbxo44 in both female and male samples. Only the female Fbxo44 KD showed a significant decrease in STING compared to the control.

In conclusion, FBXO44 deletion impairs contractile function and induces hypertrophic gene expression in NRVMs, however there was no increase in repetitive elements or DNA damage markers. Our ongoing studies are focused on uncovering the sex-specific role of FBXO44 in the heart. We will continue investigating histological changes including cardiomyocyte size. We will perform qPCR analysis of heart tissue to support our in vitro qPCR results in NRVMs. From this project I learned echocardiography, qPCR, and Western Blot protocols.

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