A stem cell-based model of the human muscle spindle for studying proprioceptive dysfunction in distal arthrogryposis syndromes

点击翻译按钮获取中文摘要

PROJECT SUMMARY Distal arthrogryposis (DA) syndromes are a collection of congenital disorders characterized by joint contractures and orofacial dysmorphisms. The most common genetic cause of DA phenotypes are autosomal dominant missense mutations in the MYH3 gene, encoding the embryonic myosin heavy chain. It has been suggested that expression of mutant MYH3 within contractile muscle fibers is responsible for the developmental defects that characterize DA pathology. However, the contribution of intrafusal fibers to the etiology of DA phenotypes has not yet been investigated. Intrafusal fibers are specialized cells of the muscle spindle; a proprioceptive structure responsible for regulating contractile activity in response to stretch. Since rapid tissue growth during embryogenesis leads to dynamic changes in mechanical cues throughout the organism, it seems logical to assume that defects in spindle function could severely impact the ability for the musculature to respond correctly to these signals. This, coupled with the fact that MYH3 expression persists in intrafusal fibers past embryonic stages of development, highlight the importance of studying the contribution of intrafusal fibers to DA pathology. One of the reasons that intrafusal fibers have not been studied in relation to DA syndromes is the scarcity of spindle structures in normal muscle. Only about 50,000 spindles are present in the entire human musculature, making in extremely unlikely that one will be present within a given biopsy sample. To overcome this issue, we will utilize induced pluripotent stem cells (iPSCs) to produce human intrafusal fibers with both normal and MYH3 mutant genotypes. In Aim 1, optimization of these cells from iPSCs will be performed using primary rodent tissue as a benchmark for spindle morphology. Optimized iPSC-derived spindle cells will then be subjected to controlled stretch to quantify their activation in response to mechanical cues and to characterize any functional differences that arise between mutant and control cells. As it is not yet known whether mechano-sensitive ion channels are present in the intrafusal fiber membrane or in the membrane of associated sensory neurons (or both), these experiments will be conducted in isolation and in co-culture with type 1a sensory neurons. In Aim 2, iPSC-derived intrafusal fibers will be subjected to single cell RNA sequencing to characterize the transcriptome of MYH3 mutant and normal spindle cells and identify whether the expression of mutant MYH3 contributes to an altered phenotype that persists to later stages of development. Again, primary rodent cells will be used to establish a benchmark transcriptomic signature for spindle cell types. Results from iPSC-derived intrafusal cells will be compared to those obtained from mutant and wild type extrafusal (contractile) muscle fibers to determine whether the transcriptomic impact of mutant MYH3 is more pronounced in the spindle than in the surrounding cells of the musculature. Overall, this project will increase our understanding of intrafusal fiber biology, provide a new in vitro assay for probing spindle function, and help determine whether mutant intrafusal fibers contribute to DA etiology.