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Toward the use of whole, live developing zebrafish as models for skeletal and cardiac muscle contraction

Martin, Brit Leigh, Martin
http://orcid.org/0000-0003-4889-4990
http://rave.ohiolink.edu/etdc/view?acc_num=osu1500611447591388

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
The basic unit of contractile machinery in cardiac and skeletal muscle is the sarcomere. This array of proteins contains thick filaments made up of myosin motors that bind actin in thin filaments to generate tension. In all types of striated muscle calcium binds the C subunit of troponin on the thin filaments. When bound by calcium, troponin C initiates a series of events to remove inhibition of myosin binding sites on actin. In myopathies mutations in sarcomeric, calcium handling, or structural lead to malfunction of the muscle and disease. The gap in our understanding lies in how mutations in such proteins affect the fundamental function of muscle, and how changes in basic muscle function lead to disease. Therefore, it is important to develop new methods to distinguish between benign and pathologic mutations and their respective effects on muscle function. Many experimental paradigms are used to study how myopathy-causing mutations affect muscle. Simpler systems (like isolated myofilaments) have fewer variables but are missing the regulatory processes present in intact muscle. Mouse models maintain regulatory processes but do not always recapitulate human disease phenotypes. Zebrafish are excellent models for studying muscle physiology and function for reasons including: a manipulable genome, a large ratio of muscle to body size, and amenabilities to chemical treatment and live imaging. In addition, their small size permits stimulation of the entire trunk musculature simultaneously. I improved techniques by developing an assay to directly measure muscle strength. Many previous methods analyzed images of swimming larvae. Using anesthetized fish, I bypassed motor input and electrically stimulated trunk musculature. I developed a companion assay to measure cross-sectional area (CSA) of muscle. I characterized contractile strength in developing wild-type muscle and distinguished between muscle phenotypes in morphant zebrafish. We characterized morphants in which Rbfox1l and Rbfox2 RNA-binding protein expression was reduced. The resulting paralysis, due to a muscle-autonomous defect, was perceptible by our method. I also developed an assay to measure eccentric contractions in 3 days post fertilization (dpf) zebrafish muscle. Eccentric contractions’ damaging effects can indicate skeletal muscle’s susceptibility to injury. In characterizing wild-type muscle, I found that stretch during contraction significantly increases developed force. Optimization of this assay may indicate the zebrafish as a model for understanding the mechanisms of force increase during lengthening. I tried to develop a transient transgenic zebrafish model for muscle-specific expression of myopathy-causing mutant proteins. Due to atypical mosaicism in my initial expression experiments, I was not able to evaluate whether this transient transgenic, mosaic approach to muscular expression of human cardiac troponin C (hcTnC) isoforms is sufficient to answer questions about the effects of mutant isoforms on basic muscle function. Additional optimization is necessary. The results outlined herein add to the knowledge of zebrafish muscle physiology. I improved assays for zebrafish muscle physiology and made steps toward developing a real-time assay of properties of muscle contraction in genetic mutants. In the future, this improved methodology will streamline the investigation of myopathy mutations and will provide insights into how myopathy mutations modify muscle physiology.
Paul Janssen (Advisor)
Sharon Amacher (Committee Member)
Christine Beattie (Committee Member)
Jon Davis (Committee Member)
135 p.
Biomedical Research; Biophysics; Cellular Biology; Molecular Biology; Physiology
zebrafish, myopathy, muscle contraction, striated muscle, rbfox, cardiac muscle, skeletal muscle

Recommended Citations

Citations

  • Martin, Martin, B. L. (2017). Toward the use of whole, live developing zebrafish as models for skeletal and cardiac muscle contraction [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1500611447591388

    APA Style (7th edition)

  • Martin, Martin, Brit. Toward the use of whole, live developing zebrafish as models for skeletal and cardiac muscle contraction. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1500611447591388.

    MLA Style (8th edition)

  • Martin, Martin, Brit. "Toward the use of whole, live developing zebrafish as models for skeletal and cardiac muscle contraction." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1500611447591388

    Chicago Manual of Style (17th edition)

osu1500611447591388
172
© 2017, some rights reserved.Creative Commons License Toward the use of whole, live developing zebrafish as models for skeletal and cardiac muscle contraction by Brit Leigh Martin Martin is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.
Release 3.2.12