Transcriptome reprogramming is a key process of pathological remodeling in heart. In mammalian transcriptome, a significant portion of the genes produce more than one transcript species due to alternative RNA splicing. However, the contribution of alternative RNA splicing to total transcriptome complexity and its regulatory mechanism in heart failure is still poorly understood and understudied. Based on RNA sequencing and extensive validation studies, we have discovered that global pattern of alternative RNA splicing adapts a fetal-like profile in diseased hearts, including a highly conserved mutually exclusive splicing for all members of the transcriptional factor Mef2 family, Mef2a, Mef2c and Mef2d. We further find that Fox1 is a muscle enriched trans-acting RNA splicing factor that regulates this specific Mef2 splicing event in heart, producing splicing variants with distinct transcriptional activities and different functional impact in heart. Fox1 expression is diminished in mouse and human failing hearts. Inactivation of Fox1 in zebrafish causes developmental defects and cardiac dysfunction. Most remarkably, restoring Fox1 expression in mice significantly attenuates cardiac hypertrophy and dysfunction induced by pressure-overload. Therefore, alternative RNA splicing is a highly regulated process that significantly contributes to the transcriptome programming in heart. It has an important and previously underappreciated impact on cardiac development and pathogenesis. These exciting new findings lead to our current hypothesis that Fox1-MEF2 is a novel regulatory circuit in cardiac transcriptional network with a pivotal role in heart failure. In this proposal, we will expore this novel hypothesis at molecular and functional levels in multiple model systems in order to fully establish the underling mechanism and the functional importance of Fox-1 mediated RNA splicing regulation in heart failure. More specifically, we plan to accomplish in Aim 1: to determine the specific contribution of Fox-1 to transcriptome complexity in cardiomyocytes; in Aim 2: to investigate the molecular basis and functional impact of Mef2a splicing variants in heart; in Aim 3: to establish the functional impact of Fox-1-Mef2 circuit in cardiac hypertrophy and heart failure. These studies will provide exciting new insights to cardiac transcriptome regulation in normal development and diseases, and promising new targets for therapeutic development.