The Role of Limch1 Alternative Splicing in Skeletal Muscle Function

点击翻译按钮获取中文摘要

Project Summary The goal of this project is to determine the functional consequences of developmentally regulated and conserved LIM and calponin homology domain 1 (Limch1) alternative splicing in skeletal muscle. Postnatal muscle development is a highly dynamic period associated with extensive transcriptome remodeling. A significant aspect of this process is widespread changes in alternative splicing, required for adaptation of tissues to adult function. The functional significance of many developmental and tissue specific alternative splicing transitions is unknown. These splicing events have significant implications since reversion of adult mRNA isoforms to fetal isoforms is observed in many skeletal muscle diseases. Limch1 is a putative actin-binding protein with LIM and calponin homology domains and several protein isoforms of unknown significance, generated by alternative splicing. The Limch1 gene expresses a ubiquitous protein isoform (uLimch1) in most tissues and a skeletal muscle specific isoform that predominates in adult skeletal muscle (mLimch1). mLimch1 contains an additional internal and in frame 454 amino acids encoded by six contiguous exons simultaneously included after birth. The developmental regulation and tissue specificity of this splicing transition is conserved in mouse and human. However, the significance of including these six exons of Limch1 in adult muscle is yet to be determined. To determine the physiologically relevant functions of mLimch1 and uLimch1 isoforms, CRISPR-Cas9 was used to delete the genomic segment containing the 6 alternatively spliced exons of Limch1 in vivo, thereby forcing the constitutive expression of the predominantly fetal isoform, uLimch1 in adult skeletal muscle (HOM Limch1 6exKO). Preliminary grip strength analysis showed that adult male and female mice from two independent homozygous (HOM) Limch1 6exKO founder lines had significant muscle weakness in vivo while maximum force and rate of relaxation is impacted ex vivo compared to wild-type (WT) age-matched controls. I will use the HOM Limch1 6exKO mice to determine the contribution of the muscle specific Limch1 isoform to skeletal muscle physiology and function. In the first aim, I will conduct extensive analysis of the HOM Limch1 6exKO mice through grip strength testing to determine extent of progression with aging, a thorough panel of ex vivo contractility assays to identify the predominant mechanism affecting force generation and skeletal muscle calcium analysis to identify the physiological defects resulting from removal of mLimch1, the Limch1 isoform normally present in adult muscle. In the second aim, functional characteristics of Limch1 protein will be delineated through in vivo localization studies and identification of interacting protein partners to determine the underlying mechanisms leading to strength loss in HOM Limch1 6exKO mice. By investigating the functional, spatial, and protein binding characteristics of mLimch1, the role of tissue specific regulation of Limch1 in skeletal muscle homeostasis will be uncovered. The results obtained from these studies will contribute to our understanding of the significance of RNA processing in skeletal muscle development and disease.