Differential Splicing of the Large Sarcomeric Proteins Titin and Nebulin is Developmentally Regulated and is Altered in Genetically Engineered Mice

Persistent Link:
http://hdl.handle.net/10150/323451
Title:
Differential Splicing of the Large Sarcomeric Proteins Titin and Nebulin is Developmentally Regulated and is Altered in Genetically Engineered Mice
Author:
Buck, Danielle Elizabeth
Issue Date:
2014
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Skeletal muscle is composed of repeating units called sarcomeres which contain distinct sets of thin and thick filaments that slide past each other during contraction. In addition to these proteins a third filament called titin acts as a molecular spring and prevents overstretching of muscle. In skeletal muscle, titin's spring-like elements include the PEVK sequence which elongates upon stretch and immunoglobulin-like (Ig) domains. A fourth myofilament, nebulin, is anchored into the Z-disk and present along the length of the thin filament. Nebulin is proposed to be a regulator of thin filament length. These large sarcomeric proteins can be differentially spliced to yield proteins of various sizes and properties. During my graduate career I sought to elucidate the role of titin and nebulin in skeletal muscle development and disease. Firstly, I studied if differential splicing of titin and nebulin occurred during development. Early post-natal development is a time of rapid isoform switching and growth, and I hypothesized that these large proteins could be affected. In post-natal development of the mouse, large compliant titin molecules are gradually replaced with shorter, stiffer isoforms through removal of PEVK exons. In nebulin, C-terminal exons present in the Z-disk are differentially expressed between muscle types and throughout development which correlates with differences in Z-disk width. My research has shown that titin and nebulin transcripts are tuned during development with changes in titin affecting the I-band region of the molecule and changes in nebulin affecting the Z-disk region. Secondly, I sought to study the effect of specific titin domains on titin elasticity in skeletal muscle. Changes in titin's stiffness occur in various myopathies but whether these are a cause or an effect of the disease is unknown. To test this, a genetically engineered mouse model was created in which part of the constitutively expressed immunoglobulin-like (Ig) domains of titin (Ig3-11) were removed. Unexpectedly, the deletion of these domains causes additional differential splicing to take place in skeletal muscle and leads to skeletal muscle myopathy. I sought to investigate the mechanism by which this occurs and found that RBM20, a titin splice factor, was significantly increased in IG KO mice and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Through the use of this model the mechanisms that underlie titin alternative splicing were explored and demonstrated how alternative splicing alters muscle function. My third project was to better understand the mechanisms by which nebulin loss causes the disease nemaline myopathy (NM). We generated a mouse model in which nebulin's exon 55 is deleted (NEBΔex55) to replicate a founder mutation seen frequently in NM patients with Ashkenazi Jewish heritage. The mice phenocopy pathology of severe myopathy with a short lifespan, changes in thin filament length and cross bridge cycling kinetics, and changes in calcium sensitivity. Force generation in this model is improved by the addition of a calcium sensitizer and supports the use of these compounds in treating patients with nemaline myopathy. In my last project, a second mouse model in which nebulin levels are reduced (NEB cKO) was created to study the effects of reduced nebulin levels on survival and pathology of skeletal muscle. NEB cKO mice recapitulate many of the hallmark features of typical congenital NM, and mice have muscle type dependent effects on contractility and trophicity. Most notably, the intact EDL muscle had a 84% reduction in maximal active force compared to that of the soleus muscle which had a 42% reduction in force. These differences can be explained in part by changes in thin filament length, cross bridge cycling kinetics, and muscle fiber disarray. In conclusion, through the use of genetically engineered mouse models, differential splicing of titin and nebulin during development has been characterized and mechanisms by which mutations in these large sarcomeric proteins cause skeletal muscle disease have been elucidated.
Type:
text; Electronic Dissertation
Keywords:
Molecular & Cellular Biology
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Molecular & Cellular Biology
Degree Grantor:
University of Arizona
Advisor:
Granzier, Henk

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleDifferential Splicing of the Large Sarcomeric Proteins Titin and Nebulin is Developmentally Regulated and is Altered in Genetically Engineered Miceen_US
dc.creatorBuck, Danielle Elizabethen_US
dc.contributor.authorBuck, Danielle Elizabethen_US
dc.date.issued2014-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractSkeletal muscle is composed of repeating units called sarcomeres which contain distinct sets of thin and thick filaments that slide past each other during contraction. In addition to these proteins a third filament called titin acts as a molecular spring and prevents overstretching of muscle. In skeletal muscle, titin's spring-like elements include the PEVK sequence which elongates upon stretch and immunoglobulin-like (Ig) domains. A fourth myofilament, nebulin, is anchored into the Z-disk and present along the length of the thin filament. Nebulin is proposed to be a regulator of thin filament length. These large sarcomeric proteins can be differentially spliced to yield proteins of various sizes and properties. During my graduate career I sought to elucidate the role of titin and nebulin in skeletal muscle development and disease. Firstly, I studied if differential splicing of titin and nebulin occurred during development. Early post-natal development is a time of rapid isoform switching and growth, and I hypothesized that these large proteins could be affected. In post-natal development of the mouse, large compliant titin molecules are gradually replaced with shorter, stiffer isoforms through removal of PEVK exons. In nebulin, C-terminal exons present in the Z-disk are differentially expressed between muscle types and throughout development which correlates with differences in Z-disk width. My research has shown that titin and nebulin transcripts are tuned during development with changes in titin affecting the I-band region of the molecule and changes in nebulin affecting the Z-disk region. Secondly, I sought to study the effect of specific titin domains on titin elasticity in skeletal muscle. Changes in titin's stiffness occur in various myopathies but whether these are a cause or an effect of the disease is unknown. To test this, a genetically engineered mouse model was created in which part of the constitutively expressed immunoglobulin-like (Ig) domains of titin (Ig3-11) were removed. Unexpectedly, the deletion of these domains causes additional differential splicing to take place in skeletal muscle and leads to skeletal muscle myopathy. I sought to investigate the mechanism by which this occurs and found that RBM20, a titin splice factor, was significantly increased in IG KO mice and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Through the use of this model the mechanisms that underlie titin alternative splicing were explored and demonstrated how alternative splicing alters muscle function. My third project was to better understand the mechanisms by which nebulin loss causes the disease nemaline myopathy (NM). We generated a mouse model in which nebulin's exon 55 is deleted (NEBΔex55) to replicate a founder mutation seen frequently in NM patients with Ashkenazi Jewish heritage. The mice phenocopy pathology of severe myopathy with a short lifespan, changes in thin filament length and cross bridge cycling kinetics, and changes in calcium sensitivity. Force generation in this model is improved by the addition of a calcium sensitizer and supports the use of these compounds in treating patients with nemaline myopathy. In my last project, a second mouse model in which nebulin levels are reduced (NEB cKO) was created to study the effects of reduced nebulin levels on survival and pathology of skeletal muscle. NEB cKO mice recapitulate many of the hallmark features of typical congenital NM, and mice have muscle type dependent effects on contractility and trophicity. Most notably, the intact EDL muscle had a 84% reduction in maximal active force compared to that of the soleus muscle which had a 42% reduction in force. These differences can be explained in part by changes in thin filament length, cross bridge cycling kinetics, and muscle fiber disarray. In conclusion, through the use of genetically engineered mouse models, differential splicing of titin and nebulin during development has been characterized and mechanisms by which mutations in these large sarcomeric proteins cause skeletal muscle disease have been elucidated.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectMolecular & Cellular Biologyen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMolecular & Cellular Biologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGranzier, Henken_US
dc.contributor.committeememberGranzier, Henken_US
dc.contributor.committeememberGregorio, Carolen_US
dc.contributor.committeememberKrieg, Paulen_US
dc.contributor.committeememberTsao, Tsu-Shuenen_US
dc.contributor.committeememberKonhilas, Johnen_US
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