Identification and Function of MEF2 Protein Networks in Cardiac Muscle

Studies reported herein are focused on cardiomyocyte biology to uncover mechanisms involved in the regulation of the transcription in cardiac muscle cells. In particular, the activity of MEF2 transcription factors, previously found to be dynamically modulated through post-translational modifications and protein interactions in cardiac gene expression, was a focus. Thus, exploring the protein-protein networks that regulate the MEF2A transcription factor was a primary goal in these studies. To address this, a systematic unbiased screen of the MEF2 interactome has been carried out in primary cardiomyocytes (PCMs) using a quantitative proteomic analysis approach based on isobaric labelling using iTRAC followed by LC-qMS/MS. In these experiments, we identified novel regulatory MEF2A protein partners, by which may help to explain the diverse roles that MEF2 exerts in the control of cardiomyocyte gene expression. In particular, we documented STAT3 protein complexed with MEF2A, the interaction was subsequently confirmed by different biochemical assays. While some studies have reported a non-genomic prosurvival function of STAT3 in mitochondria, less is known about the role of STAT3 as a transcription factor in cardiomyocytes. In order to assess this interaction with MEF2A in cardiomyocytes, we performed both Gain- and Loss Of Function (GOF/LOF) studies of STAT3. GOF and LOF were achieved by overexpression (GOF) or siRNA depletion (LOF) of STAT3, respectively. We found that STAT3 exhibited an inhibitory effect on MEF2-dependent transactivation properties on synthetic (4xMEF2 Luc) and natural promoter (⍺-MHC Luc) based reporter genes. In addition, we used transcriptome analysis of siRNA depletion of STAT3 in cardiomyocytes coupled with bioinformatic analysis to identify differentially regulated genes and the cellular processes that they might regulate. These studies highlighted a potentially protective, pro-survival role of STAT3 in cardiomyocytes. Also, comparative transcriptome profiling of STAT3 and MEF2A target genes in cardiomyocytes was performed to identify common downstream target genes and potentially common functions between the two transcription factors. We also employed an integrative informatic approach using different types of omics data using previously performed MEF2A ChIP-seq and MEF2A siRNA data along with the newly identified MEF2A interactome to identify functional nodes within the data and to build predicted causal relationships for future analysis.

The signaling mechanism of the β-adrenergic receptor system (β-AR) in the heart is an important regulator in both physiological and pathological conditions. Previously, myocardial MEF2 was identified as a target of β-adrenergic signaling that modulates its transcriptional regulatory properties at its pro-survival target genes. In the second part of this thesis, we used transcriptomic analysis to assess the global changes in transcript expression in response to Isoproterenol (ISO) treatment, a β-adrenergic agonist. Transcriptome analysis in these studies revealed extensive remodeling of the cardiomyocyte transcriptome, which suggested dynamic modulation of Wnt and G-protein coupled receptor signaling, calcium handling, and potassium ion transmembrane transport in cardiomyocytes. Then, we analyzed remodeling of the chromatin landscape using ATAC-seq analysis in order to determine the response to ISO treatment. ATAC analysis identified 287 differential regions in response to the activation of β-adrenergic signaling by ISO. Comparative analysis between RNA-seq and ATAC-seq indicated that ISO may alter gene expression by modulating the chromatin landscape and therefore accessibility to transcription regulators. In addition, we compared these data with previously determined MEF2A ChIP-seq and siMEF2A RNA-seq results. This analysis suggests that ISO treatment modulates MEF2A transactivation at genes that regulate several different processes in cardiomyocytes, such as adhesion junction, proliferation, and transmembrane ion transport. Collectively, studies reported in this thesis support the multifunctional role of MEF2A in cardiomyocytes and its dependence on protein interactions and signal pathway integration.