Poster abstracts

Poster number 12 submitted by Twishasri Dasgupta

Gene expression analyses of MHC-CELFΔ mice implicate CELF-mediated alternative splicing in contractile gene regulation

Twishasri Dasgupta (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic), Andrea N. Ladd (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic)

Abstract:
Members of the CUGBP and ETR3 like factor (CELF) protein family regulate alternative splicing in the developing heart. The requirement for CELF splicing activity in heart muscle has been demonstrated with MHC-CELFΔ transgenic mice. In this model, CELF activity is inhibited in the heart muscle postnatally via the expression of a nuclear dominant negative protein (NLS-CELFΔ) under an α-MHC-promoter. MHC-CELFΔ mice develop cardiomyopathy characterized by alternative splicing defects, enlarged hearts and severe contractile dysfunction. A higher expressing “severe” line develops a severe form of cardiomyopathy, while a lower expressing “mild” line exhibits a milder form of the phenotype. Interestingly, both cardiac function and size recovers with age in the mild line without the loss of NLS-CELFΔ or change in the endogenous CELF protein expression. The severe line does not recover, perhaps because of extensive, irreversible muscle loss within the first few weeks.
In this study, expression profiles of wild type and MHC-CELFΔ hearts at 3 weeks were compared using gene expression microarrays. Gene ontology and pathway analyses identified calcium signaling and contraction as the most affected processes. Differences in expression levels were validated for several contractile and calcium regulating genes by real time RT-PCR and Western blot. To determine whether gene expression changes recover in the mild line with function as the animals age, we compared wild type and transgenic mice at 24 weeks. Even though cardiac function returns to normal in the mild line, surprisingly few transcripts return to wild type levels of expression and most remain affected. This suggests an additional compensatory pathway is activated. Additional array experiments are being performed with 24 week-old mice to compare the global expression patterns in wild type versus transgenic to elucidate these compensatory pathways.

Keywords: CELF proteins, Alternative splicing, Transgenic mice