Members of the CELF family of RNA binding proteins have been implicated in alternative splicing regulation in developing heart. Transgenic mice that express a nuclear dominant-negative CELF protein specifically in the heart (MHC-CELF⌬) develop cardiac hypertrophy and dilated cardiomyopathy with defects in alternative splicing beginning as early as 3 weeks after birth. MHC-CELF⌬ mice exhibit extensive cardiac fibrosis, severe cardiac dysfunction, and premature death. Interestingly, the penetrance of the phenotype is greater in females than in males despite similar levels of dominant-negative expression, suggesting that there is sexspecific modulation of splicing activity. The cardiac defects in MHC-CELF⌬ mice are directly attributable to reduced levels of CELF activity, as crossing these mice with mice overexpressing CUG-BP1, a wild-type CELF protein, rescues defects in alternative splicing, the severity and incidence of cardiac hypertrophy, and survival. We conclude that CELF protein activity is required for normal alternative splicing in the heart in vivo and that normal CELF-mediated alternative splicing regulation is in turn required for normal cardiac function.The importance of the posttranscriptional regulation of gene expression has been driven home in the last few years, as it is now known that the vast number of proteins thought to comprise the human proteome are generated from a much smaller number of genes (16). Pre-mRNA alternative splicing is an important mechanism for the generation of protein diversity (14). It is currently estimated that 60 to 74% of human genes undergo alternative splicing (17,18). Tight regulation of alternative splicing is necessary for the appropriate temporal and spatial control of gene expression, and disruption of splicing regulation can cause or contribute to human disease (11).Members of the CUG binding protein (CUG-BP) and the embryonic lethal abnormal vision type RNA binding protein 3 (ETR-3)-like factor (CELF) family of RNA binding proteins (also called BRUNOL proteins) regulate alternative splicing by binding to intronic elements within specific pre-mRNA targets (7,10,19,21,35,38). Two CELF proteins, CUG-BP1 (also known as BRUNOL2) and ETR-3 (also known as BRUNOL3, CUG-BP2, or NAPOR), are expressed in the heart and are hypothesized to drive changes in alternative splicing during cardiac development (22). CUG-BP1 and ETR-3 have also been suggested to play key roles in the pathogenesis of a number of disorders in heart and skeletal muscle, including myotonic dystrophy, Duchenne and Becker muscular dystrophies, partial monosomy 10p, and familial arrhythmogenic right ventricular dysplasia (7,23,24,35,38,40). These reports suggest an important role for CELF-mediated alternative splicing regulation in the myocardium.We previously described CELF⌬, a truncated form of CELF4 (also known as BRUNOL4) that acts as a dominant negative to specifically repress the alternative splicing activity of the members of the CELF family in cultured cells without globally disrupting alternativ...