Abstract
Members of the CUG-BP, Elav-like family (CELF) regulate alternative splicing in the heart. In MHC-CELFΔ transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an α-myosin heavy chain promoter. MHC-CELFΔ mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELFΔ mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF) network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELFΔ mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELFΔ males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor.
Highlights
The availability of complete genome sequences and high throughput sequencing data sets has revealed that alternative splicing is an important mechanism for generating diversity from a relatively limited number of genes [1,2]
The MHC-CELFD phenotype can be attributed to loss of CELF activity and not exogenous protein expression, because crossing MHC-CELFD-10 mice with lines of MCKCUG-BP1 transgenic mice that mildly over-express CELF1 in the heart results in improved alternative splicing and reduced cardiac pathogenesis in bitransgenic offspring [10]
Transgenic Mice Both MHC-CELFD-10 and MHC-CELFD-574 lines of transgenic mice were maintained as hemizygotes, so wild type littermates were used for sex- and age-matched controls
Summary
The availability of complete genome sequences and high throughput sequencing data sets has revealed that alternative splicing is an important mechanism for generating diversity from a relatively limited number of genes [1,2]. MHC-CELFD transgenic mice express a nuclear dominant negative CELF protein (NLSCELFD) in postnatal heart muscle under the control of the mouse a-myosin heavy chain promoter [10] These mice have specific defects in CELFmediated alternative splicing, and exhibit cardiac hypertrophy, dilated cardiomyopathy, severe cardiac dysfunction, and in some cases premature death [10,11]. There are two lines of MHCCELFD mice that express different levels of NLSCELFD protein: MHC-CELFD-10 (‘‘severe’’ line) mice express higher levels and MHC-CELFD-574 (‘‘mild’’ line) mice express lower levels [10,11] Both lines display dysregulation of the alternative splicing of CELF-regulated transcripts and develop cardiomyopathy, though the severe line shows a greater degree of splicing dysregulation and pathogenesis than the mild line [10,11]. Studies with this model have implicated appropriate CELFmediated alternative splicing is critical for healthy heart function, but the underlying basis of cardiomyopathy in MHC-CELFD mice remains unclear
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