RNA regulation provides a critical mechanism for controlling gene expression in the heart during normal development and disease, yet this mechanism is surprisingly understudied. The RNA-binding Fragile X (FraX) protein, FXR1, has recently been linked to proper muscle structure and cardiac hypertrophy in mouse and zebrafish, in addition to human. To decipher the direct role and mechanisms of FraX protein in the heart in vivo, we turned to the genetic capabilities of the Drosophila model, which expresses only one, functionally conserved FraX protein (dFMR1). Our long-term goal is to elucidate the components and molecular mechanisms underlying RNA regulation during normal heart development and disease. This proposal is designed to test the hypothesis that FraX proteins are required during development to regulate the structural integrity and functional properties of the heart by controlling the expression of specific cytoskeletal mRNAs. We propose the following Aims:
Aim 1 is to establish a Drosophila model of heart disease by analysis of functional and structural defects in dFMR1 mutants. Analysis of cardiac function and heartbeat parameters that include contraction strength and pacemaker activity will be measured using video microscopy and semi-automated optical heartbeat analysis software. Structural defects will be analyzed by using extensive immunofluorescence microscopy and histology to analyze muscle defects and hallmarks of cardiomyopathies. Preliminary results indicate loss of dFMR1 significantly decreases heart rate and perturbs sarcomere structure;expression of human FXR1 partially rescues this defect, supporting functional conservation between species.
Aim 2 is to identify important mRNA targets of dFMR1 in the heart to understand their molecular role(s) in heart disease. We will test the hypothesis that translational regulation by dFMR1 on its mRNA targets that encode integral cytoskeleton and cytoskeleton-associated proteins is critical for proper heart function. We propose to use RNA-IP, quantitative RT-PCR, western blot analysis and luciferase assays to identify dFMR1 mRNA targets and use genetic rescue experiments to determine their functional significance in the heart. Preliminary results indicate loss of dFMR1 decreases the expression levels of the cytoskeletal proteins talin and ZO-1, which are both targets of mammalian FXR1. The innovative approach of combining our knowledge of the powerful genetic capabilities of Drosophila with mouse and human studies concurrently being performed in the Gregorio laboratory, allow us to directly and specifically analyze the functional properties of a FraX protein in the heart. Taken together, these integrative experiments will establish the role of FraX proteins and the mechanisms by which they regulate mRNA expression during heart development and disease.
The mechanisms regulating cardiac muscle structure by RNA expression are necessary in the heart during development and in response to stress, yet they are surprisingly understudied. Here we propose to take a combined molecular and genetic approach to decipher the role of the Fragile X family of RNA binding proteins in cardiac development and disease. Our work is likely to uncover novel insights into the normal mechanisms of heart development and may provide therapeutic targets for cardiac hypertrophy.
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