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Structural and Functional Roles of Nebulin in Skeletal Muscle

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Structural and Functional Roles of Nebulin in Skeletal Muscle UNIVERSITY OF CALIFORNIA, SAN DIEGO STRUCTURAL AND FUNCTIONAL ROLES OF NEBULIN IN SKELETAL MUSCLE A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Bioengineering by David Samuel Gokhin Committee in charge: Professor Richard L. Lieber, Chair Professor Robert L. Sah, Co-Chair Professor Ju Chen Professor Andrew D. McCulloch Professor Jeffrey H. Omens 2009 i Copyright David Samuel Gokhin, 2009 All rights reserved. ii The dissertation of David Samuel Gokhin is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _________________________________________________ _________________________________________________ _________________________________________________ _________________________________________________ Co-Chair _________________________________________________ Chair University of California, San Diego 2009 iii This dissertation is dedicated to my mother, Liya, whom I lost far too soon. You were proud of me no matter what. I miss you tons and think about you everyday. Love you, Ma. iv TABLE OF CONTENTS Signature Page.…………………………………………………………….............. iii Dedication.…………………………………………………………………............. iv Table of Contents.………………………………………………………………….. v List of Abbreviations and Symbols.………………………………………………... viii List of Figures and Tables.…………………………………………………………. ix Acknowledgements.………………………………………………………………... xi Vita.………………………………………………………………………………… xiv Abstract of the Dissertation……..…………………………………………............. xvi Chapter 1 Skeletal Muscle Physiology and the Role of Nebulin …...……………. 1 1.1 General Introduction to the Dissertation.………………………………. 1 1.2 Basic Principles of Skeletal Myogenesis.……………………………… 4 1.3 Basic Principles of Skeletal Muscle Mechanics.………………............. 6 1.4 Myosin in Skeletal Muscle Physiology.……………………………….. 8 1.5 Intermediate Filaments in Skeletal Muscle Physiology.……………….. 9 1.6 Titin in Skeletal Muscle Physiology.…………………………………... 12 1.7 Mechanisms of Thin Filament Length Regulation.……………………. 13 1.8 Nebulin: Structure and Function.………………………………………. 14 1.9 The Nebulin-Knockout Mouse.………………………………………... 16 1.10 Clinical Relevance of Nebulin.……………………………………….. 18 1.11 References.……………………………………………………………. 18 Chapter 2 Quantitative Analysis of Neonatal Skeletal Muscle Functional Improve- ment in the Mouse.………………………………………………................. 33 2.1 Summary.………………………………………………………………. 33 2.2 Introduction.……………………………………………………………. 34 2.3 Materials and Methods.………………………………………………… 36 2.3.1 Animals and Experimental Design.………………………….. 36 2.3.2 Measurement of Fiber Cross-Sectional Area.………………... 36 2.3.3 Measurement of Area Fraction of Contractile Material.……... 37 2.3.4 Muscle Functional Assessment.……………………………… 38 2.3.5 Muscle Architecture and Isometric Stress Calculation.…...…. 39 2.3.6 Validation of Muscle Functional Assessment.……………….. 40 2.3.7 Gel Electrophoresis of Myosin Heavy Chain Isoforms.……... 40 v 2.3.8 Western Blotting for Desmin.………………………………... 41 2.3.9 Statistics.……………………………………………………... 41 2.4 Results.…………………………………………………………………. 42 2.5 Discussion.……………………………………………………………... 45 2.6 Acknowledgments.……………………………………………………... 49 2.7 References.…………………………………………………………....... 49 Chapter 3 Reduced Thin Filament Length in Nebulin-Knockout Skeletal Muscle Al- ters Isometric Contractile Properties…………………….…………………. 58 3.1 Summary.………………………………………………………………. 58 3.2 Introduction…………………………………………………………….. 59 3.3 Materials and Methods…………………………………………………. 62 3.3.1 Animals and Experimental Model …………………………... 62 3.3.2 Muscle Architecture and Isometric Stress Measurement ……. 63 3.3.3 Muscle Functional Assessments …………………………….. 65 3.3.4 Sarcomere Length Measurements …………………………… 66 3.3.5 Sarcolipin RNA Expression Analysis ……………………….. 68 3.3.6 Myosin Heavy Chain Analysis ……………………………… 69 3.3.7 Transmission Electron Microscopy …………………………. 69 3.3.8 Statistics ……………………………………………………... 70 3.4 Results………………………………………………………………….. 70 3.4.1 Evidence for Functional and Structural Muscle Deterioration. 70 3.4.2 Sarcolipin RNA Expression Analysis ……………………….. 71 3.4.3 Myosin Heavy Chain Analysis ……………………………… 72 3.4.4 Cyclic Contractile Testing …………………………………... 73 3.4.5 Length-Tension Curves and Sarcomere Length Measurement. 73 3.5 Discussion……………………………………………………………… 76 3.6 Acknowledgements…………………………………………………….. 84 3.7 References……………………………………………………………… 85 Chapter 4 Physiological Roles of the Extreme C-Terminal SH3 Domain of Nebulin in Skeletal Muscle: Implications for Nemaline Myopathy………………… 103 4.1 Summary.………………………………………………………………. 103 4.2 Introduction…………………………………………………………….. 104 4.3 Materials and Methods…………………………………………………. 106 4.3.1 Creation of Experimental Animals ………………………….. 106 4.3.2 Transmission Electron Microscopy …………………………. 108 4.3.3 Histology and Fiber Size Measurement ……………………... 108 4.3.4 Myosin Heavy Chain Isoforms ……………………………… 109 4.3.5 Measurement of Isometric Stress Production ……………….. 110 4.3.6 Eccentric Contraction-Induced Injury ……………………….. 112 4.3.7 Force-Frequency Relationship ………………………………. 113 4.3.8 Passive Mechanical Testing of Single Muscle Fibers ……….. 113 vi 4.3.9 Isometric Contraction-Induced Signaling …………………… 115 4.3.10 Statistics ……………………………………………………. 116 4.4 Results………………………………………………………………….. 116 4.4.1 Baseline Characteristics ……………………………………... 116 4.4.2 Response to Eccentric Contraction-Induced Injury …………. 117 4.4.3 Force-Frequency Relationship ………………………………. 118 4.4.4 Passive Mechanical Properties of Single Muscle Fibers ……. 119 4.4.5 Isometric Contraction-Induced Signaling …………………… 119 4.5 Discussion……………………………………………………………… 120 4.6 Acknowledgements…………………………………………………….. 125 4.7 References……………………………………………………………… 125 Chapter 5 Conclusions…...……………………………………………………….. 136 5.1 Summary and Significance of Findings.……………………………….. 136 5.2 Future Directions…………………………………………………....…. 139 5.2.1 Mechanism of Neonatal Lethality …………………………… 139 5.2.2 Manipulation of Nebulin’s Functional Domains ……………. 140 5.2.3 Gene Therapy Approaches …………………………………... 142 5.3 References……………………………………………………………… 144 vii LIST OF ABBREVIATIONS AND SYMBOLS ANOVA analysis of variance ATP adenosine triphosphate bHLH basic helix-loop-helix BSA bovine serum albumin CSAf fiber cross-sectional area D diameter Ecc# eccentric contraction # EDL extensor digitorium longus EHL extensor hallucis longus ELISA enzyme-linked immunosorbent assay EMB embryonic myosin heavy chain ERK extracellular regulated kinase FWHM full-width at half-maximum GAS gastrocnemius Ig immunoglobulin Iso# isometric contraction # JNK c-Jun N-terminal kinase Lf fiber length Lm muscle length Lopt optimum relative muscle length Ls sarcomere length 0 Ls slack sarcomere length M muscle mass MAPK mitogen-activated protein kinase MEK MAPK/ERK kinase MyHC myosin heavy chain NEO neonatal myosin heavy chain NEB-KO nebulin-knockout P# postnatal day # PCR polymerase chain reaction PCSA physiological cross-sectional area PCSA* adjusted physiological cross-sectional area PMSF phenylmethylsulfonyl fluoride PS# passive stretch # ρ muscle density SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SH3 Src homology 3 θ pennation angle TA tibialis anterior WT wild-type Xcsa cross-sectional area fraction of contractile material viii LIST OF FIGURES AND TABLES Figure 1.1. Schematic of myogenic precursor cell diversification and differentiation during skeletal myogenesis ……………………………………………....... 26 Figure 1.2. The hierarchical organization of skeletal muscle……………………… 27 Figure 1.3. Length-tension relationship from single muscle fibers in the frog …… 28 Figure 1.4. Force-velocity relationship from single muscle fibers in the frog ……. 29 Figure 1.5. Schematic of skeletal muscle cytoarchitecture ………………………... 30 Figure 1.6. Models of thin filament length specification in striated muscle ……… 31 Figure 1.7. The molecular structure of one isoform of mouse nebulin …………… 32 Figure 2.1. Photograph of the experimental apparatus for functional testing of mouse pup hindlimbs ……………………………………………………………... 53 Figure 2.2. Laminin immunohistochemistry and phalloidin staining of transverse TA muscle sections ……………………………………………………………. 54 Figure 2.3. Postnatal time-courses of body mass, muscle architecture, and isometric stress ……………………………………………………………………….. 55 Figure 2.4. Postnatal time-courses of myosin heavy chain isoform levels ………... 56 Figure 2.5. Postnatal time-course of desmin levels ……………………………….. 57 Figure 3.1. Images of the muscle-testing chamber for isometric testing of the neonatal mouse GAS ………………………………………………………………... 90 Figure 3.2. Sample stress-time traces of WT and NEB-KO GAS at P1 during isometric activation ………………………………………………………... 91 Figure 3.3. Isometric stress production in the GAS muscle of WT and NEB-KO mice at P1 and P7 and its relationship to muscle ultrastructure ………………… 92 Figure 3.4. Relative quantification of sarcolipin RNA transcript levels in WT and NEB-KO tibialis anterior muscle tissue from P1 to P11 ………………….. 93 Figure 3.5. Myosin heavy chain isoform distributions in the WT and NEB-KO GAS at P1 and P7 ……………………………………………………………….. 94 ix Figure 3.6. Responses of the WT and NEB-KO GAS at P1 to cyclic isometric activation …………………………………………………………………... 95 Figure 3.7. Length-tension curves of the WT and NEB-KO GAS at P1 ………….. 96 Figure 3.8. Sarcomere length in the WT and NEB-KO GAS at P1, either at slack length or stretched by +1 Lf ………………………………………………... 97 Figure 3.9. Parabolic regression analysis of the length-tension properties of the WT and NEB-KO GAS at P1 ...…………………………………………………
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