Calpains in skeletal muscle: Generation of an inhibitory overexpression system and analysis of degradation in simulated microgravity

Persistent Link:
http://hdl.handle.net/10150/280476
Title:
Calpains in skeletal muscle: Generation of an inhibitory overexpression system and analysis of degradation in simulated microgravity
Author:
Grill, Mischala Ann
Issue Date:
2003
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:
Muscle atrophy is a serious side effect seen with extended time in space. Proteolytic degradation of specific muscle proteins leads to smaller, weaker muscles that are structurally more susceptible to damage. Calpains are proteases that specifically degrade target proteins of the myofibril, and have been implicated in many types of muscle atrophy. Calpain activity is regulated by a combination of activation by calcium and inhibition by calpastatin, its endogenous inhibitor. This dissertation describes the generation of a skeletal muscle-specific, doxycycline (Dox) controlled, calpastatin over-expression system in transgenic mice to regulate calpain activity. A dual construct system, the transactivator line utilizes an optimized tet-on system and a modified muscle creatine kinase promoter to create muscle specific expression of a tet transactivator. The second transgenic line, consisting of a bi-directional promoter centered on a tet responder element controlling both a luciferase reporter gene and a tagged calpastatin, is transcriptionally silent until activated by a dox induced transactivator molecule. Compound hemizygous mice showed high level, Dox dependent, muscle-specific overexpression of luciferase and transgenic calpastatin, demonstrating the effectiveness and flexibility of the tet-on system to provide a tightly regulated overexpression system in adult skeletal muscle. Consistent overexpression of calpastatin was hard to maintain, however, and not all of the proposed experiments could be achieved. Additional studies compared the degradation of hindlimb suspended mouse muscle proteins (weightlessness model) to those of ground control muscle proteins briefly incubated in Ca²⁺ (to initiate calpain degradation). Four proteins known as targets for calpain degradation were selected for analysis. Degradation responses of myofibrillar proteins titin, nebulin, and troponin T in hindlimb suspension clearly mimicked that seen with the calcium incubations. The cytoskeletal calpain target protein, desmin, however did not respond the same to both treatments showing moderate degradation with calcium and no degradation with hindlimb suspension. These data suggest that myofibrillar calpain target proteins, but not necessarily cytoskeletal proteins, are rapidly targeted for degradation in hindlimb suspension in a manner similar to that induced by calcium, implicating calpain as a mediator of this degradation.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Genetics.; Biology, Cell.; Biology, Animal Physiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Cell Biology and Anatomy
Degree Grantor:
University of Arizona
Advisor:
Antin, Parker B.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleCalpains in skeletal muscle: Generation of an inhibitory overexpression system and analysis of degradation in simulated microgravityen_US
dc.creatorGrill, Mischala Annen_US
dc.contributor.authorGrill, Mischala Annen_US
dc.date.issued2003en_US
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.abstractMuscle atrophy is a serious side effect seen with extended time in space. Proteolytic degradation of specific muscle proteins leads to smaller, weaker muscles that are structurally more susceptible to damage. Calpains are proteases that specifically degrade target proteins of the myofibril, and have been implicated in many types of muscle atrophy. Calpain activity is regulated by a combination of activation by calcium and inhibition by calpastatin, its endogenous inhibitor. This dissertation describes the generation of a skeletal muscle-specific, doxycycline (Dox) controlled, calpastatin over-expression system in transgenic mice to regulate calpain activity. A dual construct system, the transactivator line utilizes an optimized tet-on system and a modified muscle creatine kinase promoter to create muscle specific expression of a tet transactivator. The second transgenic line, consisting of a bi-directional promoter centered on a tet responder element controlling both a luciferase reporter gene and a tagged calpastatin, is transcriptionally silent until activated by a dox induced transactivator molecule. Compound hemizygous mice showed high level, Dox dependent, muscle-specific overexpression of luciferase and transgenic calpastatin, demonstrating the effectiveness and flexibility of the tet-on system to provide a tightly regulated overexpression system in adult skeletal muscle. Consistent overexpression of calpastatin was hard to maintain, however, and not all of the proposed experiments could be achieved. Additional studies compared the degradation of hindlimb suspended mouse muscle proteins (weightlessness model) to those of ground control muscle proteins briefly incubated in Ca²⁺ (to initiate calpain degradation). Four proteins known as targets for calpain degradation were selected for analysis. Degradation responses of myofibrillar proteins titin, nebulin, and troponin T in hindlimb suspension clearly mimicked that seen with the calcium incubations. The cytoskeletal calpain target protein, desmin, however did not respond the same to both treatments showing moderate degradation with calcium and no degradation with hindlimb suspension. These data suggest that myofibrillar calpain target proteins, but not necessarily cytoskeletal proteins, are rapidly targeted for degradation in hindlimb suspension in a manner similar to that induced by calcium, implicating calpain as a mediator of this degradation.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Genetics.en_US
dc.subjectBiology, Cell.en_US
dc.subjectBiology, Animal Physiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineCell Biology and Anatomyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorAntin, Parker B.en_US
dc.identifier.proquest3119949en_US
dc.identifier.bibrecord.b45631827en_US
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