The proper function of cardiac muscle requires the highly orchestrated assembly of thousands of cytoskeletal and regulatory proteins into individual sarcomeres (contractile units) and membrane-associated junctional complexes. A critical, yet extremely understudied, aspect of this highly regulated process is the requirement for mRNA localization and translation at the site of protein function. The long-term goal of this research is to identify the molecular mechanisms governing mRNA regulation during cardiac development and hypertrophy. In this proposal, we focus on the role of the RNA binding protein FXR1, the striated muscle- specific member of the Fragile X protein family, in controlling the expression of specific mRNAs in cardiac muscle. Using a combined cellular, molecular and genetic approach we obtained extensive preliminary data that identified the first mRNA targets of FXR1 in the heart. These include mRNAs that encode proteins associated with specialized cytoskeletal assemblies such as costameres (talin) and intercalated discs (desmoplakin). Remarkably, electron and confocal microscopy revealed severe structural perturbations in junctional complexes that are consistent with dysregulation of the mRNA targets we identified, and provide a plausible explanation for the cause of the FXR1 knockout (KO) embryonic lethality. Preliminary results also showed that FXR1 protein (and its direct targets that we identified) is misregulated identically in mouse and human cardiac hypertrophy, and that FXR1 KO hearts exhibits numerous molecular signatures of heart disease. Our results together with current models for the function of Fragile X family members form the basis for our hypothesis that FXR1 regulates cardiac muscle development and function by controlling the localization, stability and/or translation of specific mRNA targets encoding essential cytoskeletal proteins during normal and pathological conditions. Using mouse heart tissue and primary cardiomyocytes, we aim to: 1) determine the phenotypic consequences of FXR1 loss in KO hearts using extensive high-resolution immunofluorescence and electron microscopy. These experiments will be complemented by siRNA knock-down coupled with live-cell imaging in primary cardiac myocytes; 2) identify mRNA targets of, and determine the manner in which they are regulated by FXR1 using both candidate and genome-wide approaches. State-of-the-art molecular methods will be utilized to determine whether FXR1 directly binds its mRNA targets and to establish the mode of regulation (localization, stability and/or translation of the mRNA); and 3) decipher the role of FXR1 in cardiac hypertrophy/stress and identify its novel hypertrophy-specific targets. Several methods described in Aim 2 will be used in conjunction with mouse models of cardiac hypertrophy and functional assessment of cardiac muscle physiology. With this integrative approach, we are well positioned to decipher the molecular mechanisms utilized by FXR1 during de novo cardiac muscle assembly and myopathy/stress. To our knowledge we are the only group investigating this critical aspect of muscle development. Furthermore, the discovery of mRNA targets regulated by FXR1 during normal development and in diseased states may identify novel therapeutic approaches for heart disease.
The mechanisms regulating RNA localization and expression are critical for heart muscle development and in disease, yet they are remarkably understudied. Here we propose to take a combined cellular, molecular and genetic approach to decipher the role of the RNA binding protein FXR1, a member of the Fragile X family of proteins, in cardiac development and hypertrophic stress. Identification of novel cardiac FXR1 mRNA targets will likely uncover insights into therapeutic targets for cardiomyopathy.
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