The congenital myopathies are a diverse group of inherited neuromuscular disorders that manifest as skeletal muscle weakness at birth or in infancy, and are classically defined by the predominant morphological features observed on muscle biopsy. The goals of this dissertation were to better understand the pathophysiology behind these devastating diseases and to identify new therapeutic approaches through the use of faithful vertebrate models.
Due to their proliferative capacity, transparency, and well-characterized genome, zebrafish represent a robust vertebrate model system to study muscle development. In the first part of this work, we created and characterized a novel zebrafish model of centronuclear myopathy using antisense morpholinos targeting the bridging integrator 1 (bin1) gene. Bin1 morphant skeletal muscles revealed structural defects reported in human biopsies, and live calcium imaging offered new mechanistic insights linking abnormal triads to impairments in intracellular signaling.
Later studies focused on two forms of core myopathy, and utilized stable zebrafish models to guide development of targeted and effective therapies. We began by using TALE nucleases to generate germ line mutations in the zebrafish selenoprotein N (sepn1) gene, and in doing so created the first vertebrate to accurately model human SEPN1-related myopathy (SEPN1-RM). Sepn1 zebrafish mutants exhibited morphological abnormalities, reduced contractile strength, and skeletal muscle “cores” under electron microscopy. We then showed that the sepn1 phenotype could be ameliorated by pharmacological inhibition of a thiol oxidase localized at the sarcoplasmic reticulum. These data served as the first in vivo evidence to indicate that reactive oxygen species significantly contribute to SEPN1-RM, and may do so by impairing calcium re-uptake following muscle contraction. Finally, we performed a medium-throughput chemical screen on the closely related relatively relaxed (ryr1b) zebrafish, and identified JAK-STAT cytokine signaling as a druggable molecular pathway relevant to these pathologies.
In summary, these studies increase our knowledge of the affected systems in both centronuclear and core myopathies, and provide strong in vivo support that these conditions arise from defects in skeletal muscle excitation-contraction coupling. This work also further establishes zebrafish-based small molecule screens as a powerful tool for lead compound identification and drug development in human genetic disease. / Medical Sciences
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/23845448 |
| Date | 04 December 2015 |
| Creators | Smith, Laura Lindsay |
| Contributors | Cepko, Constance, Harris, Matthew, Bonnemann, Carsten, MacRae, Calum, Beggs, Alan |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
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