YTHDF3 as a critical regulator of cardiac function

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PROJECT SUMMARY As the global leading cause of death, heart failure is a major challenge for researchers in their quest to discover therapeutics that can save countless lives. After cardiac injury, the heart begins to remodel itself in a way that is initially adaptive, but this innate coping mechanism may over time expedite heart failure onset. Elucidating the mechanisms which underly the progression from adaptive cardiac hypertrophic remodeling to heart failure will dramatically impact the discovery of novel therapeutics for this deadly disease. While regulation of gene expression through transcription of messenger RNA (mRNA) has been extensively studied, only recently an appreciation for the importance of chemical modifications that can occur on mRNA has emerged. This proposal focuses on the methylation of the N6-Adenosine of mRNA (m6A), which is the most abundant internal mRNA modification in eukaryotes. Previous research from our lab has shown that modulation of m6A content in the heart is sufficient to drive cardiac remodeling and to affect the ability of the heart to respond to stress. Despite this, the exact mechanisms through which this occurs is not well understood. The fate of m6A-modified mRNAs is regulated by members of the YTH Domain Family (YTHDF). We found that YTHDF3 is specifically important in cardiomyocytes, where it localizes to the nucleus and binds to Myocyte Enhancer Factor 2D (MEF2D), which is an important transcription factor regulating hypertrophic cardiac growth. Further, we have found that knockout of YTHDF3 mitigates pathological remodeling following pressure overload injury. Given these preliminary data, we hypothesize that YTHDF3 regulates cardiomyocyte size and stress-induced remodeling by modulating the processing of m6A-modified mRNAs transcribed by MEF2D. To test this hypothesis, we already generated and validated a new mouse line in which YTHDF3 can be selectively deleted in cardiomyocytes (YTHDF3-cKO). In Aim 1, we will investigate the role of YTHDF3 at baseline and in the stressed murine heart using longitudinal echocardiography analysis, and assessing histological and molecular signs of pathology at the terminal time point. In Aim 2, we will determine the mechanism through which YTHDF3 regulates the fate of specific subsets of MEF2D-transcribed m6A-mRNAs in cardiomyocytes. First, we will further characterize the binding between YTHDF3 and MEF2D by defining the respective domains involved. Then, we will dissect the binding of YTHDF3 to MEF2D mRNA targets and determine consequent stability, export, and translation of these transcripts. Finally, in Aim 3, we will undertake an unbiased approach to more globally investigate the role of YTHDF3 in regulating mRNA biology in healthy and stressed adult cardiomyocytes by cross-linking immunoprecipitations of YTHDF3-bound mRNAs followed by sequencing (CLIP-seq). Our approach is innovative and significant, as it will be the first project to define the role of YTHDF3 in the heart, which may lead to a new field of therapeutics based on the biology of mRNA methylation.