Titin Splicing Mechanisms and Physical Implications

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DESCRIPTION (provided by applicant): Titin is an extremely large protein found primarily in cardiac and skeletal muscle. It is alternatively spliced and different splice isoforms affect the function of the protein. We have partially characterized a mutation in rats that dramatically alters the splicing pattern of titin in the heart. The mutated gene responsible for altered titin splicing has been localized to an RNA binding protein in chromosome 1. Transfection of adenovirus constructs contain the Rbm20 sequence into cultured cardiomyocytes from homozygous mutants will be used in rescue experiments. Binding of the Rbm20 to RNA or DNA will be explored using ChIP assays. Alternative mechanisms whereby titin splicing is developmentally changed will be explored. These include developmental up regulation of Rbm20 message and protein using quantitative PCR and Western blotting respectively, developmental alterations in Rbm20 phosphorylation state, or developmental changes in other protein or proteins that associate with Rbm20. The role of titin in stretch dependent signal transduction will be explored using the titin splice mutation model for comparisons with wild type animals after pressure and volume overload challenges. Experiments where the titin stretch signals can be balance using heterozygote mutants and a model system where titin expression is altered by propylthiouricil will be conducted. Changes in gene expression will be measured using quantitative PCR and Western blots. Studies will be conducted to characterize the prolonged duration of action potentials from cardiomyocytes of homozygous mutants and determine whether the altered ion channel properties found in isolated cells are also observed in vivo. We will also use the mutant rat model to explore titin's role in length dependent activation and the Frank-Starling relationship using isolated skinned cardiomyocytes. X- ray diffraction experiments using skinned trabeculae will compare filament spacing of wild type, heterozygote, and homozygote mutants at different sarcomere lengths. The studies outlined should provide new insights into titin function and the mechanisms controlling its isoform expression. They may also help to better understand the role of ion channel alternative splicing on cardiac arrhythmias. PUBLIC HEALTH RELEVANCE: Titin isoforms changes have been shown to occur in dilated cardiomyopathy, so understanding the mechanisms controlling titin splicing and the cardiac adaptations to different titin isoforms may help in devising treatments for this prevalent cardiovascular disease. In addition the rat mutation model we have developed should help to understand mechanisms of cardiac arrhythmia and sudden death.