Mitochondrial-Sarcoplasmic Reticulum Crosstalk:

Mitochondrial Subcellular Localization, Calcium Uptake, and

Suppression of Local Calcium Release in Fast-Twitch Skeletal Muscle

During Postnatal Development

By

Ann Elizabeth Rossi

Submitted in Partial Fulfillment

of the

Requirements for the Degree

Doctor of Philosophy

Supervised by

Professor Robert T. Dirksen

Department of Pharmacology & Physiology

School of Medicine and Dentistry

University of Rochester

Rochester, NY

2009
 ii

DEDICATION

To my parents, for free dinners, clean laundry, and most importantly, their

unwavering support and encouragement.

 iii

CURRICULUM VITAE

The author was born in Elmira, New York on March 4, 1980. She attended

Nazareth College of Rochester from 1998 to 2002, where she received the Dean’s

Scholar Award. In 2001, she held an internship with Corning Incorporated in the

NMR group of the Characterization Science and Services Department, researching

different formulations of polymer coatings for optical fibers. In 2002, she graduated

Magna Cum Laude with a Bachelor of Science degree in Biology. Shortly thereafter,

in the Fall of 2002, she began graduate studies at the University of Rochester School

of Medicine and Dentistry. She pursued her research in Pharmacology under the

direction of Professor Robert T. Dirksen and received the Master of Science degree

from the University of Rochester in 2005. In 2006, she was awarded a Biophysical

Society Student Travel Grant to support her attendance and presentation at the 50th

Annual Biophysical Society Meeting. From 2006 to 2008, she received additional

training and financial support from a National Institute of Dental and Craniofacial

Research Training Grant. Following graduation, the author will be working as a

Postdoctoral Scholar for Dr. Elizabeth McNally at the University of Chicago in

Chicago, Illinois.

PUBLICATIONS

Rossi AE, Dirksen RT. Sarcoplasmic Reticulum: The Dynamic Calcium Governor of

Muscle. Muscle & Nerve 2006;33(6):715-731.

 iv

Durham WJ*, Aracena-Parks P*, Long C*, Rossi AE, Goonasekera SA,

Boncompagni S, Gilman CP, Galvan DL, Baker M, Shirokova N, Protasi F, Dirksen

RT, Hamilton SL. RyR1 S-Nitrosylation Underlies Environmental Heat Stroke and

Sudden Death in Y522S RyR1 Knock-in Mice. Cell 2008;133(1):53-65.

Rossi AE, Boncompagni S, Dirksen RT. Sarcoplasmic Reticulum-Mitochondrial

Symbiosis: Bidirectional Signaling in Skeletal Muscle. Exercise and Sport Sciences

Reviews 2009;37(1):29-35.

Boncompagni S, Rossi AE, Micaroni M, Beznoussenko GV, Polishchuk RS, Dirksen

RT, Protasi F. Mitochondria are Linked to Calcium Stores in Striated Muscle by

Developmentally Regulated Tethering Structures. Molecular Biology of the Cell,

2009;20(3):1058-67.

* Indicates co-first authorship.

 v

ACKNOWLEDGEMENTS

There are several deserving individuals who I would like to thank for making

my graduate career rewarding, both academically and personally. First, I thank my

advisor Dr. Robert T. Dirksen for allowing me the freedom to pursue research outside

of the lab “box” and pioneer new techniques in the laboratory. His mentoring has

prepared me well to meet the challenges of a post-doctoral position and a future

career in research, which I appreciate more than I can express. Furthermore, I extend

my thanks to the members of my thesis committee: Dr. Paul. Brookes, Dr. Shey-

Shing Sheu, and Dr. David Yule, for helpful input and guidance on my project,

especially in prioritizing experiments.

Several collaborators provided necessary materials and scientific input on this

research. I express my gratitude to Dr. Werner Melzer at the University of Ulm for

kindly allowing me to use the automated detection software for calcium spark

detection. Also, I would like to thank Dr. Susan Hamilton at Baylor College of

Medicine for the knock-in mice and for graciously hosting my visit to her laboratory.

Moreover, my gratitude goes to Dr. Clara Franzini-Armstrong at the University of

Pennsylvania for helpful technical discussions and sharing her expertise. Most

importantly, I thank Drs. Simona Boncompagni and Feliciano Protasi at the Gabriele

d'Annunzio University for their fruitful collaboration. I am particularly appreciative

of Simona’s time and effort on the electron microscopy analysis, and for allowing me

to present some of her work. Additionally, this research would not have been possible

without the financial support from the National Institutes of Health Grants AR044657
 vi

and 5P01AR052354 and National Institute of Dental and Craniofacial Research

Training Grant T32DE07202.

Furthermore, my graduate career could not have been as productive or

enjoyable without the members of the Dirksen laboratory. I extend my thanks to

Linda Groom and Sara Geitner for all they do, day-to-day, to keep the laboratory

running. In particular, I thank Linda for helping to pass the time between

experiments, exchanging notes about the previous night’s reality TV shows. Also, I

am grateful to all current and former Dirksen laboratory members for their productive

scientific discussions, in particular, Dr. John Lueck, for his support and interest in

both my work and my success in science.

In addition, I would like to thank all of my friends for their companionship,

for sharing frustrations and successes in research, and for reminding me to relax and

have fun. Finally, I am grateful to my entire family for their love and support

throughout my graduate career and my life, especially my sisters Julie and Johanna,

who provided the perfect balance of coddling and “telling-it-like-it-is”, and to my

parents for teaching me the value of hard work and the power of prayer and positive

thinking. Thank you for your encouragement. Most of all, to my twin, Marie, thank

you for being my toughest critic, my biggest fan, and my best friend.
 vii

ABSTRACT

In adult skeletal muscle, mitochondria are primarily located next to calcium

release units (CRUs), or triads, the structures formed by the close apposition of

sarcoplasmic reticulum (SR) terminal cisternae and transverse tubules (T-tubules).

Close SR-mitochondria colocalization and the formation of functional calcium

microdomains permit rapid mitochondrial calcium uptake during physiological

elevations in myoplasmic calcium. At the same time, exclusive positioning of

mitochondria adjacent to CRUs is a prerequisite for inhibition of local calcium release

(i.e. Ca2+ sparks). In this way, a structural association of mitochondria with the triad

provides a basis for privileged bidirectional SR-mitochondrial communication

between the two organelles.

However, limited information is available with regard to mitochondrial

disposition during skeletal muscle development. To this end, the following research

characterized mitochondrial localization in flexor digitorum brevis (FDB) fibers from

young (0.5-1 month of age) and adult (2-4 months of age) mice using both confocal

and electron microscopy. As expected, adult FDB fibers exhibited mitochondrial

staining similar to that of SR proteins located at the SR-T-tubule junction, consistent

with mitochondrial localization to CRUs. Additionally, imaging studies revealed a

progression of mitochondrial localization from longitudinal clusters to an exclusively

junctional position from 2 weeks to 4 months. Electron microscopy analysis

confirmed the increase in mitochondrion-CRU colocalization throughout postnatal

muscle development. Collectively, the data indicates that mitochondrion-CRU

 viii

targeting is a highly coordinated and developmentally regulated process and suggests that

mitochondrial calcium uptake and function are altered during muscle development.

To determine how mitochondrial calcium uptake varied with mitochondrion-

CRU targeting, mitochondrial calcium uptake was measured in FDB fibers from

young (1 month of age) and adult (4 months of age) mice by confocal imaging of

Rhod-2 fluorescence. In response to tetanic stimulation trains, calcium accumulation

in triadic and longitudinal mitochondria was significant in fibers isolated from both 1

and 4 month-old mice. The increase in mitochondrial free calcium persisted for the

duration of tetanic stimulation and returned to resting levels within twenty minutes

after stimulation. Interestingly, the global calcium chelator EGTA had varied effects

on the increase in mitochondrial calcium. Specifically, EGTA reduced mitochondrial

calcium uptake in 4 month triadic mitochondria. Taken together, these results suggest

that mitochondrial calcium uptake was permitted by the global calcium and provide

evidence for SR-mitochondrial calcium signaling.

Additionally, to investigate the influence of mitochondria on local calcium

release, the incidence and properties of calcium sparks, revealed by exposure to

hypertonic Ringer, were measured with confocal line-scanning at different times

during postnatal skeletal muscle development (0.5-4 months). Previous studies

correlating mitochondrial content and metabolic capacity with the development of

calcium sparks in permeabilized skeletal muscle fibers concluded that actively

respiring mitochondria inhibit calcium sparks. In a similar fashion, mitochondrial

localization adjacent to CRUs facilitated inhibition of local calcium release at the

 ix

CRU. Specifically, calcium spark frequency decreased, concurrent with an increase

in mitochondrion-CRU pairing. The results are consistent with a role for

mitochondria in suppressing local calcium release in mammalian skeletal muscle.

Presumably, retrograde and orthograde communication between SR and

mitochondria is necessary to maintain normal calcium homeostasis and cellular

metabolism in skeletal muscle. Therefore, a breakdown in the bidirectional signaling

originating from the SR, mitochondria, and/or the structural interaction between them

could lead to muscle dysfunction and disease. Accordingly, the final goal of this

work was to characterize mitochondrial localization and function in skeletal muscle

from a mouse model of the muscle disorders Malignant Hyperthermia (MH) and

Central Core Disease (CCD). Surprisingly, no gross changes in mitochondrial

localization were detected with confocal imaging. Relative to wild-type muscle,

mitochondria in knock-in mouse fibers followed the same progression during

postnatal development from relatively random longitudinal clusters to precise

positioning at the triad. However, electron microscopy analysis revealed focal

regions of swollen and damaged mitochondria intimating that the aberrant SR

calcium release, characteristic of these mice, initiates pathological changes in

mitochondria.

Copyright © 2009 Ann Elizabeth Rossi

 x

TABLE OF CONTENTS

DEDICATION .............................................................................................................. ii

CURRICULUM VITAE .............................................................................................. iii

ACKNOWLEDGEMENTS .......................................................................................... v

ABSTRACT ................................................................................................................ vii

TABLE OF CONTENTS .............................................................................................. x

LIST OF TABLES ..................................................................................................... xiv

LIST OF FIGURES .................................................................................................... xv

ABBREVIATIONS .................................................................................................. xvii

FOREWORD .............................................................................................................. xx

INTRODUCTION ........................................................................................................ 1

I.1. SARCOPLASMIC RETICULUM (SR)............................................................. 2

I.1.1. Calcium Storage .......................................................................................... 3

I.1.2. Calcium Release ........................................................................................ 11

I.1.3. Calcium Reuptake...................................................................................... 23

I.2. MITOCHONDRIA ........................................................................................... 27

I.2.1. Subcellular Localization in Muscle ........................................................... 27

I.2.2. Oxidative Phosphorylation (OXPHOS)..................................................... 29

I.2.3. Mitochondrial Calcium Uptake ................................................................. 33

I.2.4. Mitochondrial Inhibition of Local Calcium Release ................................. 35

I.3. MUSCLE DYSFUNCTION AND DISEASE.................................................. 38

I.3.1. Fatigue ....................................................................................................... 38

 xi

I.3.2. Exercise-Induced Muscle Damage ............................................................ 40

I.3.3. Malignant Hyperthermia............................................................................ 42

I.3.4. Central Core Disease ................................................................................. 44

I.3.5. Brody Disease ............................................................................................ 46

I.4. OVERVIEW ..................................................................................................... 48

CHAPTER 1: MITOCHONDRIAL SUBCELLULAR LOCALIZATION ............... 57

1.1. RATIONALE................................................................................................... 58

1.2. METHODS ...................................................................................................... 58

1.2.1. Preparation of Muscle Fibers .................................................................... 58

1.2.2. Confocal Imaging and Analysis of Mitochondrial Localization and

Membrane Potential ................................................................................ 59

1.2.3. Electron Microscopy (EM) ....................................................................... 61

1.3. RESULTS ........................................................................................................ 62

1.3.1. Mitochondria-CRU Colocalization Increases During Postnatal

Development ........................................................................................... 62

1.3.2. JC-1 Emission Distribution and Magnitude are Altered During Postnatal

Development ........................................................................................... 63

1.4. DISCUSSION .................................................................................................. 64

CHAPTER 2: MITOCHONDRIAL CALCIUM UPTAKE ....................................... 74

2.1 RATIONALE.................................................................................................... 75

2.2. METHODS ...................................................................................................... 75

2.2.1. Preparation of Muscle Fibers .................................................................... 75

 xii

2.2.2. Measurements of Myoplasmic Calcium ................................................... 76

2.2.3. Confocal Imaging and Analysis of Mitochondrial Calcium Uptake ........ 76

2.3. RESULTS ........................................................................................................ 78

2.3.1. Mitochondria Accumulate Calcium Following Tetanic Stimulation ........ 78

2.3.2. EGTA Reduces Mitochondrial Calcium Uptake During Tetanic

Stimulation .............................................................................................. 82

2.4. DISCUSSION .................................................................................................. 84

CHAPTER 3: MITOCHONDRIAL SUPPRESSION OF LOCAL CALCIUM

RELEASE ....................................................................................................... 94

3.1. RATIONALE................................................................................................... 95

3.2. METHODS ...................................................................................................... 95

3.2.1. Preparation of Muscle Fibers .................................................................... 95

3.2.2. Confocal Imaging and Analysis of Calcium Sparks ................................. 96

3.3. RESULTS ........................................................................................................ 97

3.3.1. Spatiotemporal Properties of Osmotic Shock-Induced Calcium Sparks

Vary in an Age-Dependent Manner. ....................................................... 97

3.3.2. Osmotic Shock Induces Subsarcolemmal Changes ................................ 100

3.4. DISCUSSION ................................................................................................ 101

CHAPTER 4: MITOCHONDRIAL FUNCTION IN SKELETAL MUSCLE

DISEASE ...................................................................................................... 116

4.1. RATIONALE................................................................................................. 117

4.2. METHODS .................................................................................................... 117

 xiii

4.2.1. Preparation of Muscle Fibers .................................................................. 117

4.2.2. Confocal Imaging and Analysis of Mitochondrial Localization and

Membrane Potential .............................................................................. 118

4.2.3. Calculation of Mitochondrial Spacing .................................................... 118

4.2.4. Time Lapse Imaging and Analysis of Superoxide Flashes ..................... 119

4.3. RESULTS ...................................................................................................... 120

4.3.1. JC-1 Emission Magnitude is Altered During Postnatal Development in

Y522S Knock-in Mice .......................................................................... 120

4.3.2. Mitochondrial Spacing is Similar in WT and YS FDB Fibers .............. 121

4.3.3. Focal Mitochondrial Damage in YS FDB Fibers ................................... 122

4.4. DISCUSSION ................................................................................................ 123

CONCLUSIONS....................................................................................................... 131

BIBLIOGRAPHY ..................................................................................................... 137

 xiv

LIST OF TABLES

Table 1.1. Quantitative Analysis of CRUs and Mitochondria ................................... 69

Table 3.1. Calcium Spark Spatiotemporal Properties .............................................. 109

Table 3.2. Electron Microscopy Analysis of Subsarcolemmal Mitochondria ......... 110

 xv

LIST OF FIGURES

Figure I.1. Structural Features of Key Calcium Regulatory Proteins of the Skeletal

Muscle Sarcoplasmic Reticulum..................................................................... 52

Figure I.2. Location and Interactions of the Different SR Calcium Regulatory

Proteins Within One Side of the Triad ............................................................ 53

Figure I.3. Mitochondria are Precisely Localized Adjacent to the Calcium Release

Unit in Adult Mammalian Fast-Twitch Skeletal Muscle ................................ 54

Figure I.4. Schematic Representation of Interactions Between Components of the

Mitochondrial Calcium Transport Systems, the Tricarboxylic Acid (TCA)

Cycle, and the Electron Transport Chain (ETC) ............................................. 55

Figure I.5. Bidirectional SR-Mitochondrial Signaling in Skeletal Muscle ................ 56

Figure 1.1. Mitochondria-CRU Colocalization Increases During Postnatal

Development ................................................................................................... 70

Figure 1.2. Electron Microscopy (EM) Analysis of Increased Mitochondrion-CRU

Positioning ...................................................................................................... 71

Figure 1.3. Mitochondrial Distribution and Membrane Potential are Altered During

Postnatal Development ................................................................................... 73

Figure 2.1. Rhod-2 Fluorescence in FDB Skeletal Muscle Fibers ............................ 89

Figure 2.2. Mag-Fluo-4 Measurements of Myoplasmic Calcium.............................. 90

Figure 2.3 Mitochondrial Membrane Potential is Stable During Tetanic

Stimulation ...................................................................................................... 91

Figure 2.4. Mitochondrial Calcium Increases During Tetanic Stimulation ............... 93

 xvi

Figure 3.1. Osmotic Shock-Induced Ca2+ Sparks and Bursts in FDB Fibers During

Postnatal Development ................................................................................. 111

Figure 3.2. Temporal Properties of Osmotic Shock-Induced Calcium Sparks Vary in

an Age-Dependent Manner ........................................................................... 112

Figure 3.3. Ca2+ Spark Amplitude Peaks at 1 Month During Postnatal Development . 113

Figure 3.4. Osmotic Shock-Induced Ca2+ Spark Frequency per CRU Density

Decreases with Age....................................................................................... 114

Figure 3.5. Osmotic Shock Induces Swelling of T-Tubules and Caveolae ............. 115

Figure 4.1 JC-1 Emission Ratio is Altered During Postnatal Development in Y522S

Knock-In Mice .............................................................................................. 127

Figure 4.2. Mitochondrial Disposition and Sarcomere Spacing in Adult FDB Fibers . 128

Figure 4.3. Mitochondrial Ultrastructure is Altered in FDB Fibers of Y522S Knock-

In Mice .......................................................................................................... 129

Figure 4.4. Mitochondrial Superoxide Flashes in FDB Fibers ................................ 130

 xvii

ABBREVIATIONS

ADP, adenosine diphosphate

-AM, acetoxymethyl ester

AP, action potential

ATP, adenosine triphosphate

BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

CaUP, calcium uniporter

CCD, central core disease

CICR, calcium-induced calcium release

CrP, creatine phosphate

CRU, calcium release unit

DH, dehydrogenase

DHPR, dihydropyridine receptor

Di-8-ANEPPS, aminonaphthylethenylpyridinium dye

EC coupling, exitation-contraction coupling

EDL, extensor digitorum longus

EGTA, glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid

EM, electron microscopy

F, fluorescence

FADH2, reduced flavin adenine dinucleotide

FCCP, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone

FDB, flexor digitorum brevis

 xviii

FDHM, full duration at half-maximal amplitude

FKBP, FK-506 binding protein

FWHM, full width at half-maximal amplitude

IMM, inner mitochondrial membrane

mAKAP, muscle A-kinase anchoring protein)

MgATP, magnesium ATP

MH, malignant hyperthermia

MHS, malignant hyperthermia susceptible

mRyR, mitochondrial ryanodine receptor

MTG, MitoTracker® Green FM

MTR, MitoTracker® Red CMXRos

NAD+, nicotinamide adenine dinucleotide

NADH, reduced nicotinamide adenine dinucleotide

nNOS, neuronal nitric oxide synthase

NO•, nitric oxide

OMM, outer mitochondrial membrane

OXPHOS, oxidative phosphorylation

PMCA, plasma membrane calcium ATPase

RaM, rapid calcium uptake mode

RNS, reactive nitrogen species

ROS, reactive oxygen species

RyR1, ryanodine receptor type 1

 xix

SD, standard deviation

SE, standard error

SERCA, sacro(endo)plasmic reticulum calcium ATPase

SOD, superoxide dismutase

SR, sarcoplasmic reticulum

TCA, tricarboxylic acid cycle

TMRE, tetramethylrhodamine ethyl ester

TT or T-tubule, transverse tubule

VICR, voltage-induced calcium release

WT, wild-type

YS, Y522S RyR1 mutation

 xx

FOREWORD

Parts of this document contain previously published text and figures.

Portions of the text from the Introduction, as well as Figures I.1 and I.2, were

reprinted with permission of John Wiley & Sons, Inc. from the following

publication (outlined at http://www.wiley.com/WileyCDA/Section/id-

301703.html):

Rossi AE, Dirksen, RT. Sarcoplasmic Reticulum: The Dynamic Calcium

Governor of Muscle. Muscle & Nerve 2006;33(6):715-731.

Additional text from the Introduction and Figures I.3, I.4, and I.5 were

reproduced from the following publication with copyright permission granted by

Lippincott Williams & Wilkins (outlined at http://www.lww.com/resources/

permissions/journals.html):

Rossi AE, Boncompagni S, Dirksen RT. Sarcoplasmic Reticulum-

Mitochondrial Symbiosis: Bidirectional Signaling in Skeletal Muscle.

Exercise and Sport Sciences Reviews 2009;37(1):29-35.

 xxi

Chapters 1, 2, 3, and 4 represent original research completed by the

author. Figures 1.1 and 1.3 were adapted from the following publication with

copyright permission granted by the American Society for Cell Biology (outlined

at http://www.molbiolcell.org/misc/ifora.shtml):

Boncompagni S, Rossi AE, Micaroni M, Beznoussenko GV, Polishchuk

RS, Dirksen RT, Protasi F. Mitochondria are Linked to Calcium Stores in

Striated Muscle by Developmentally Regulated Tethering Structures.

Molecular Biology of the Cell 2009;20(3):1058-67.

Collaborators provided supporting data for the Introduction and Chapters 1, 3,

and 4. The data in Figures I.3B, I.3C, 1.2 and 3.5 and Tables 1.1 and 3.2 were

provided by Dr. Simona Boncompagni at the Interuniversity Institute of Myology,

Center for Excellence on Ageing, Gabriele d'Annunzio University in Chieti, Italy.

Figure 4.3 was reproduced from the following publication with copyright permission

granted by Cell Press as outlined at http://www.cell.com/Permissions:

Durham WJ, Aracena-Parks P, Long C, Rossi AE, Goonasekera SA,

Boncompagni S, Gilman CP, Galvan DL, Baker M, Shirokova N, Protasi F,

Dirksen RT, Hamilton SL. RyR1 S-Nitrosylation Underlies Environmental

Heat Stroke and Sudden Death in Y522S RyR1 Knock-in Mice. Cell

2008;133(1):53-65.
 1

INTRODUCTION
 2

Skeletal muscle contraction, at the most basic level, requires calcium and

ATP, and thus, is under the control of two major organelles, the sarcoplasmic

reticulum (SR) and mitochondria. The SR is the primary regulator of muscle calcium

cycling, mediating calcium storage, release, and reuptake. Equally important are the

mitochondria, the major source of cellular ATP. Although mitochondria are adjacent

to sites of calcium release in adult skeletal muscle, SR and mitochondria are

classically regarded for their individual roles. However, close SR-mitochondrial

colocalization in adult muscle suggests that these organelles do not operate in

isolation. Therefore, the purpose of this work is to determine the degree of structural

and functional crosstalk between these two organelles.

I.1. SARCOPLASMIC RETICULUM (SR)

The governor of a gasoline or diesel engine provides a feedback mechanism to

change speed as needed and to maintain a specified speed once it is reached. In an

analogous manner, the sarcoplasmic reticulum (SR) functions as a dynamic calcium

governor in muscle that provides an automatic feedback control for altering and

maintaining myoplasmic and SR calcium levels. Interactions between terminal

regions of the SR and the transverse tubular membrane, as well as envelopment of the

myofibrils by the longitudinal SR, uniquely position this organelle for controlling

calcium release and reuptake during muscle contraction. Given its central role in

controlling calcium cycling, the SR has developed an elaborate set of calcium

regulatory proteins. These provide for a near-instantaneous release of calcium upon

 3

excitation, and a slower means of calcium reuptake and maintained calcium storage

during muscle relaxation and inactivity. Specifically, the processes of calcium

storage, release, and reuptake are balanced by the concerted action of three major

classes of SR calcium regulatory proteins: 1) luminal calcium binding proteins

(calsequestrin, histidine-rich calcium binding protein, junctate, and sarcalumenin) for

calcium storage; 2) SR calcium release channels (type 1 ryanodine receptor or RyR1

and IP3 receptors) for calcium release; and 3) sarco(endo)plasmic reticulum calcium-

ATPase (SERCA) pumps for calcium reuptake.

I.1.1. Calcium Storage

The SR is the primary calcium storage organelle in striated muscle. The

efficacy of calcium release during excitation-contraction (EC) coupling depends on

the level and calcium binding capacity of proteins within the SR. A number of

different acidic calcium binding proteins participate in calcium buffering and help

maintain SR free calcium at levels around 1 mM.93 The most important protein in

calcium buffering and storing calcium in the terminal SR is calsequestrin (Figure

I.1A), which comprises ~27% of all junctional SR protein.65 Although originally

regarded as only a luminal calcium buffer, calsequestrin is now recognized to play a

more direct role in regulating SR calcium release during skeletal muscle EC

coupling.21
 4

Calsequestrin (CSQ)

Calsequestrin was first extracted from SR vesicles in the early 1970s by

MacLennan and colleagues.158 Initial studies found that the protein is highly acidic,

hydrophobically bonded to the inside of SR vesicles, and capable of low-affinity (kD =

40 µM) and high capacity (~40 mol calcium/mol protein at pH 7.5) calcium binding.

Thus, the protein was given the name “calsequestrin” for its ability to sequester

calcium within the SR.158

Since its original isolation, two isoforms of calsequestrin (skeletal and

cardiac) have been identified and differ in the number of acidic residues in their

C-termini. Although it bears no effect on its ability to bind calcium, skeletal

calsequestrin has fewer acidic residues in its C-terminal tail than the cardiac

isoform.21 Fast- and slow-twitch skeletal muscle express the skeletal isoform,10,28,68

whereas the cardiac isoform is expressed in both cardiac muscle and slow-twitch

skeletal muscle.28 Interestingly, both isoforms of calsequestrin are present early

during fast-twitch skeletal muscle development. As development progresses,

expression of the cardiac isoform gradually decreases until it is essentially absent in

adult muscle.10,190,204 While at the protein level, the switch from the cardiac to

skeletal muscle isoform occurs during postnatal weeks 2–4.204

Synthesis of calsequestrin is controlled by both neuronal input149 and

myogenin,10 a member of the muscle regulatory factor (MRF) family of muscle

specific transcription factors.151 Once synthesized, skeletal muscle calsequestrin

becomes localized in the lumen of the terminal SR by an as yet unknown mechanism.

 5

Interestingly, it does not possess a C-terminal Lys-Asp-Glu-Leu (KDEL) ER

retention signal characteristic of other soluble SR proteins.21

Calsequestrin has three similar domains linked by short unstructured

sequences of amino acids to form a monomer with a hydrophilic core. Each domain

has a disk-like shape with a four-stranded β-sheet at its hydrophobic core and two

hydrophilic α-helices on each side of the sheet. The structure of each domain is

reminiscent of Esherichia coli thioredoxin. At turns between the β-strands,

hydrophilic side chains form crevices that are similar to the redox centers of

thioredoxin.248 This similarity to thioredoxin structure suggests that calsequestrin

may participate in redox reactions by acting as an SR chaperone protein that mediates

disulfide bond formation in oxidative protein folding.21

Approximately one-third of calsequestrin is composed of acidic residues, most

of which are in the C-terminus of the monomer. These acidic residues are important

for calcium binding. Rather than the presence of discrete calcium binding sites, pairs

of acidic residues create a net surface charge to which calcium binds.248 As calcium

binds, the hydrating water is freed in an energetically favorable reaction.136 Calcium

concentrations in the low micromolar range (10 µM) cause collapse of the monomer’s

structure. Further increases in calcium (10–100 µM) lead to dimerization, which

results in the formation of an electronegative pocket for enhanced calcium binding.

Following dimerization, additional calcium binding at higher levels of calcium

induces polymerization. Typically, at resting SR free calcium concentrations (~1

mM) calsequestrin exists as a stable polymer.248

 6

Polymers of calsequestrin are visible as electron dense material in the terminal

SR in electron micrographs of the skeletal muscle triad.89 However, calsequestrin is

not membrane bound;87 it resides in the lumen of the SR. Calsequestrin is anchored

to the junctional face of the SR membrane through interactions with junctin, triadin,

and RyR1 (Figure I.2).266 Junctin and triadin binding involves an aspartate-rich

region (amino acids 354–367) at the C-terminus of calsequestrin.210 Potential direct

interactions between calsequestrin and RyR1 have also been observed.99,106

Triadin and Junctin

Triadin was first identified in skeletal muscle (Figure I.1B).31 Although

triadin in skeletal muscle is primarily found as a 95 kDa glycoprotein embedded in

the SR membrane, a number of different triadin splice variants have also been

identified.168,230,243 The bulk of the full-length triadin protein is located in the lumen

of the SR,132 where it interacts with calsequestrin, junctin, and RyR1. Multiple Lys-

Glu-Lys-Glu (KEKE) motifs located in the luminal portion of triadin are required for

binding to calsequestrin134 and RyR1.148 Similarly, junctin is a 20 kDa protein with a

short cytosolic N-terminus, followed by a single membrane-spanning α-helix and a

long, positively charged luminal C-terminus (Figure I.1C). Like triadin, the luminal

structure of junctin binds both calsequestrin and RyR1.124 By binding to both RyR1

and calsequestrin, triadin, and junctin anchor calsequestrin and thereby, increase

calcium buffering close to sites of SR calcium release (Figure I.2). In addition, the

quaternary complex of triadin, junctin, RyR1, and calsequestrin is thought to relay

 7

information regarding SR calcium store content to RyR1,22 ultimately affecting

calcium release, and thus, skeletal muscle contractility. The means by which

calsequestrin regulates the calcium release mechanism is discussed in greater detail in

the next section.

Histidine-Rich Calcium Binding Protein (HRC)

The role of histidine-rich calcium binding protein (HRC) as a second luminal

calcium storage protein has recently been appreciated. In native SR, HRC

(699–862 amino acids) exists as a multimer of five or more subunits.222 The primary

amino acid sequence consists of three regions: 1) a conserved N-terminal signal

sequence; 2) a central acidic histidine-rich repeat region; and 3) a conserved C-

terminal cysteine-rich region (Figure I.1D).112 The central region is responsible for

both calcium binding and interactions with other junctional proteins. Specifically, the

concentration of acidic residues in the central region may provide charge on the

protein necessary for calcium binding.147 Nonetheless, HRC is less abundant in

skeletal muscle than calsequestrin, indicating that it functions only as a secondary

calcium binding protein.128

HRC also binds to triadin, perhaps signifying a greater role for this molecule

than simply increasing the number of calcium binding sites in the SR. The repeats in

the central region are necessary for the interaction of HRC with triadin.147 The

histidine-rich region contains two different types of repeats, A and B, which are

responsible for binding to the KEKE motifs in the luminal portion of triadin. In both
 8

types of repeats, a string of acidic amino acids are flanked by histidine residues. The

number of acidic residues varies between types A and B. Similarly, the number of

terminal histidine residues differs; type A ends with a single residue, whereas type B

ends with multiple histidines.114 More than 4.5 repeats are required for triadin

binding, although the types of repeats do not affect the strength of interaction. In

addition, the interaction between HRC and triadin is calcium sensitive, such that the

strongest interaction in vitro occurs in the absence of calcium.147 Thus, increases in

SR calcium content promote triadin dissociation by disrupting the multimeric

structure of HRC.222 Changes in SR calcium content could provide additional

regulation of calcium release by shifting interactions of triadin from HRC to other

triadin-binding proteins (e.g., RyR1 and calsequestrin). An area of future research

will be to determine whether the dual functions of HRC and calsequestrin (calcium

and triadin binding) are redundant, or if the two proteins exhibit unique roles in

regulating SR calcium storage and release.

Junctate

Defining specific functional roles of different SR calcium binding proteins is

further complicated by the identification of junctate, a third calcium binding

protein in the junctional SR (Figure I.1E).237 Junctate is a 298 amino acid integral SR

membrane protein (33 kDa) with structural homology to junctin. Junctate is derived

from alternative splicing of the genes that encode both junctin and aspartyl β-

hydroxylase. The initial 78 N-terminal amino acids of junctate are identical to

 9

junctin, the first 23 of which are located in the cytoplasm. After a single-pass

transmembrane domain, the rest of the protein is located in the lumen of the SR. The

C-terminal sequence of junctate is identical to the nonenzymatic C-terminus of

aspartyl β-hydroxylase. Junctate binds approximately 21 mol calcium/mol protein

with an apparent kD of 217 µM, primarily within the negatively charged luminal C-

terminus of the protein.237 Thus, like calsequestrin and HRC, junctate is a high-

capacity, moderate-affinity SR calcium binding protein and therefore contributes to

the calcium storage/release capacity of the SR. However, unlike calsequestrin and

HRC, the calcium binding sites of junctate are tethered close to sites of calcium

release by anchoring of the protein in the junctional SR membrane via the

transmembrane domain of junctin. Because mRNA transcripts of junctate are less

abundant in skeletal muscle compared with pancreas, heart, brain, and kidney, it is

theorized that junctate plays a limited role in SR calcium storage in skeletal

muscle.237

Sarcalumenin (SAR)

The concentration of the calcium binding proteins calsequestrin, HRC, and

junctate in the junctional SR serves to significantly enhance the level of high-capacity

calcium storage close to sites of calcium release. However, this does not preclude an

important role for calcium binding proteins within nonjunctional regions of the SR.

Sarcalumenin (SAR) is a high capacity (~35 mol calcium/mol protein at pH 7.5),

moderate-affinity (kD of 300 µM) calcium binding protein that is primarily located in
 10

the longitudinal SR (Figure I.1F).146 Sarcalumenin is a splice variant of an

incompletely characterized 53 kDa glycoprotein located in the lumen of the

longitudinal SR. The full-length amino acid sequence of SAR (436 acidic amino

acids) includes a calcium binding insert sandwiched between an N-terminal signal

sequence and several putative nucleotide binding motifs for P-loop-containing

ATPases/GTPases in the C-terminal region of the protein.145,262 Sarcalumenin

colocalizes with SERCA, suggesting that the protein not only contributes to calcium

buffering within the longitudinal SR, but may also influence calcium reuptake.146

These conclusions are supported by recent results obtained from SAR knockout

mice.262

To date, no mutations in the SR calcium binding proteins just discussed have

been associated with a human skeletal muscle myopathy. However, in a few families,

autosomal-recessive catecholamine-induced cardiac polymorphic ventricular

tachycardia (CPVT) is associated with mutations in cardiac calsequestrin. One

mutation involves the substitution of a negatively charged aspartic acid residue for a

histidine at position 307.138 This aspartate residue is located in the negatively charged

C-terminus of the protein, and mutation of this residue to histidine may interfere with

calcium binding. It is proposed that disruption of calcium binding to cardiac

calsequestrin by this mutation may lead to SR calcium overload, nonevoked

oscillatory SR calcium release, and subsequent triggering of arrhythmogenic early

and delayed afterdepolarizations.138 In addition, CPVT in three other families has

been linked to three different nonsense mutations that each lead to the introduction of
 11

premature stop codons in calsequestrin.193 Given the critical role of SR calcium

binding proteins in controlling SR calcium content and the regulation of SR calcium

release in skeletal muscle, it would not be surprising if future work were to identify

mutations in calsequestrin, HRC, junctate, or sarcalumenin as contributing factors in

certain forms of skeletal muscle disease.21

I.1.2. Calcium Release

EC coupling in striated muscle is the process by which an electrical signal in

the sarcolemma (i.e. an action potential) is converted into an intracellular chemical

signal (i.e. a calcium transient). This signal is then harnessed to do work by driving

actomyosin crossbridge cycling. In skeletal muscle, the electrochemical conversion

process is coordinated by a direct mechanical interaction between T-tubular

dihydropyridine receptors (DHPRs or L-type calcium channels) and RyR1s located in

the terminal cisternae of the SR.91 In adult skeletal muscle, RyR1 proteins, or feet,

are arranged in ordered arrays within the SR terminal cisternae on either side of the

transverse tubule (T-tubule). Within these RyR1 arrays, every other calcium release

channel is associated with a “tetrad” of four DHPRs in the T-tubule membrane. The

association of a T-tubule containing DHPR tetrads, flanked on either side by two

closely apposed SR terminal cisternae bearing RyR1 calcium release channels, is

referred to as the calcium release unit (CRU) or triad.90

 12

Calcium Release Unit (CRU)

The CRUs and the EC coupling apparatus in embryonic and postnatal

development of mammalian skeletal muscle are well described in the

literature.82,88,90,225 The sequence of events follows a similar pattern with some

variation in time-course between different species and skeletal muscle types.

Following myoblast fusion during myogenesis, free SR is primarily aligned at the M-

line, though its specific arrangement with respect to the myofibrils is largely

unstructured.82 The first SR associations with exterior membranes in developing

muscle, termed peripheral couplings, occur between the SR and surface

membrane.90,225 Shortly thereafter, electron dense RyR1 feet, detected in thin

sections, start to form ordered arrays in the junctional gap between the SR and

sarcolemma. Assembly of the RyR1 arrays provides the structural requirement for

the resultant clustering of DHPRs into tetrads.90,92 These peripheral junctions

represent early stages in formation of the EC coupling apparatus. At this point, clear

transverse invaginations of the sarcolemma, or T-tubules, are not readily discernable.

Rather, primitive sarcolemmal branches, beginning at the periphery of the myofiber

and exhibiting a longitudinal orientation, are the foundations of the future T-tubular

network. Subsequent projection of these sarcolemmal invaginations further into the

myofiber interior is accompanied by an increased association with the SR.

Concurrently, the nonjunctional SR becomes a tighter network, located more

exclusively at the Z-line and M-line. Further differentiation and development of

these two membrane systems results in an increase in the formation of robust SR-T-
 13

tubule junctions, which are enriched in RyR1 arrays with alternately apposed DHPR

tetrads.88,90 Finally, the formation of the fully developed triad involves reorientation

of the CRU from a longitudinal to a transverse location between myofibril bundles

along the junction between the A- and I-bands (A-I) of the sarcomere in adult

mammalian skeletal muscle.82,90

As triads rearrange from a longitudinal to transverse orientation and localize

to the A-I junction, the speed of the contractile response increases, and the time from

the start of the action potential to the beginning of recorded muscle tension

decreases.82 This illustrates that arrangement of CRUs in a specific orientation with

respect to the myofibrils contributes to the efficiency of the EC coupling process.82

Furthermore, the structure of the mature triad maintains close juxtaposition of T-

tubular DHPRs and RyR1s in the terminal SR, guaranteeing the fidelity2 of EC

coupling. Essentially, the tight triad junction ensures that each action potential

triggers calcium release via a defined signaling interaction between DHPR and RyR1

channels. In skeletal EC coupling, this signaling interaction is bidirectional.

Orthograde coupling (DHPR to RyR1) refers to the mechanism by which

depolarization of the T-tubule membrane triggers activation of RyR1-mediated SR

calcium release, while retrograde coupling (RyR1 to DHPR) reflects the influence of

RyR1 on the calcium conductance and gating properties of the DHPR.75

 14

Junctophilin

Moreover, a certain degree of control of EC coupling exists in stabilization of

the triad structure which is essential for proper coupling between the DHPR and

RyR1.120 Establishing contact between the sarcolemma and SR and subsequent

organization of these membranes into triads is thought to be facilitated by a group of

integral SR membrane proteins called junctophilins.120,135,227 Junctophilins have two

major domains: 1) an N-terminal cytoplasmic domain, which constitutes the bulk of

the protein; and 2) a C-terminal hydrophobic, single-pass transmembrane domain.

The cytoplasmic domain of junctophilin contains several novel protein interaction

elements called MORN (membrane-occupation-and-recognition-nexus) motifs, which

have the sequence Tyr-Gln/Glu-Gly-Glu/Gln-Trp-x-Asn-Gly-Lys-x-His-Gly-Tyr-Gly.

Junctophilin MORN motifs are thought to be responsible for binding to the T-tubular

membrane, possibly through interactions with specific membrane proteins or

phospholipids.227 Four separate junctophilin subtypes (JP-1, JP-2, JP-3, and JP-4)

have been identified. JP-1 and JP-2 are both expressed in skeletal muscle,227 although

the function of JP-1 has been investigated in greater detail (Figure I.1H). JP-1 is

specifically implicated in the formation of triad junctions in skeletal muscle because

electron micrographs of mouse skeletal muscle lacking JP-1 show abnormal SR

structure as well as a reduction in the number of triad junctions.120,135 Deficiency in

JP-1 contributes to disruption in the efficiency of signal conversion during EC

coupling, and thus results in reduced force generation during muscle contraction.120
 15

Junctophilin Protein-45 (JP-45)

JP-45 is a novel integral membrane protein of the SR junctional face

membrane in skeletal, but not cardiac, muscle (Figure I.1I).270 JP-45 also has a single

transmembrane domain, a 122 amino acid cytoplasmic N-terminus, and a short

luminal C-terminus. Initial studies indicated that JP-45 is phosphorylated by PKA

and interacts with both calsequestrin and the C-terminus of the DHPR α1s-subunit

(Figure I.2),4 suggesting that this protein may regulate calcium release during EC

coupling and store-operated calcium entry during tetanus and fatigue.4 In fact, more

recently JP-45 has been found to be important for functional expression of the

DHPR.5

Dihydropyridine Receptor (DHPR)

The DHPR in skeletal muscle functions both as a sarcolemmal L-type calcium

channel and as a voltage sensor that initiates calcium release through RyR1 during

EC coupling. An action potential, through stimulation of the DHPR, initiates full

activation of RyR1 for constant maximal calcium release. However, stimuli

subsequent to the initial action potential release less calcium than the first due to

calcium inactivation of calcium release. Release is reduced or terminated before

stores are fully depleted to keep constant the amount of calcium released.192 In a

sense, the DHPR can be considered the ultimate regulator of calcium release during

EC coupling.
 16

The skeletal muscle DHPR is comprised of five different subunits: α1S, β1a,

α2/δ, and γ. The pore-forming α1S-subunit contains four homologous internal repeats

(I-IV), each with six transmembrane segments (S1-S6). Loops between

transmembrane segments S5 and S6 of each repeat form the pore of the channel, and

the fourth transmembrane segment of each repeat contains a series of positive charges

involved in voltage gating.47 The coupling of DHPR activation to RyR1 channel

opening is thought to be mediated by a physical interaction between the intracellular

II-III loop of the DHPR α1S-subunit and, as yet, undefined regions of the cytosolic

aspect of RyR1.195,197,200,201,206 However, critical contributions of other regions of the

DHPR α1S-subunit46,255 and the C-terminal region of the DHPR β1a-subunit to RyR1

coupling have also been suggested.25,26,64 In addition, a potential interaction between

calmodulin binding domains located in both RyR1 and the C-terminal region of the

DHPR α1S-subunit has been reported.208

Ryanodine Receptor (RyR)

RyRs were first identified based on their high-affinity binding to the plant

alkaloid ryanodine.41,42,55 Electrophysiological characterization of RyRs revealed that

these channels are poorly selective cation channels. They possess weak selectivity

among divalent cations or among monovalent cations, and only modest selectivity for

divalent over monovalent cations. Calcium competes with Mg2+, Na+, and K+ for

pore occupancy.86 However, promiscuity in ion selectivity enables the channel to

exhibit a very high unitary conductance (~500 pS in symmetrical solutions with

 17

monovalent charge carriers150 and ~100 pS in asymmetrical solutions with divalent

charge carriers231). This high conductance permits rapid and substantial calcium

release from the terminal SR through RyR during EC coupling.

Three RyR isoforms with distinct tissue distributions have been identified:

RyR1 is primarily expressed in skeletal muscle; RyR2 is the major isoform found in

cardiac muscle; and RyR3 is found in small amounts in a wide variety of tissues.86

This work focuses on the skeletal muscle RyR isoform, RyR1 (Figure I.1G). The

skeletal muscle SR calcium release channel is a homotetramer of RyR1 protomers.

The human RyR1 protomer contains 5038 amino acids and has a molecular weight of

~565 kDa. The C-terminal one-fifth of each RyR1 protomer contains the

transmembrane segments that anchor the protein in the terminal SR. The most recent

topological model of RyR1 predicts eight transmembrane helices.78 The N-terminal

approximately four-fifths of the protein is located in the space between the SR and T-

tubule and interacts with the DHPR and a variety of other junctional regulatory

proteins.86

RyR1 channel activity is regulated by both cytosolic and luminal calcium.

Calcium release through RyR1 exhibits a bell-shaped dependence on cytosolic

calcium; low calcium (1-10 µM) activates calcium release, whereas high calcium (1-

10 mM) inhibits release.61 However, because the myoplasmic calcium concentration

rarely ever reaches millimolar levels, the effect of high calcium on RyR1 activity is

most likely manifested under physiological conditions as an inhibition of release by

cytoplasmic magnesium binding to a nonspecific low-affinity site.143 In contrast,

 18

luminal SR calcium regulates channel activity through two mechanisms: 1) a feed-

through mechanism, in which calcium released from the SR binds to the activation

and inactivation sites on the cytoplasmic face of the channel240; and 2) a luminal

calcium sensor that increases RyR1 channel activity when SR calcium reaches

millimolar levels.108 The precise mechanism by which RyR1 activity is regulated by

luminal calcium is unknown, but appears to involve both direct effects of calcium on

the channel54,216 and calcium-mediated conformational coupling through a complex

quaternary interaction between calsequestrin, junctin, triadin, and RyR1.20,22 Recent

results indicate that calsequestrin acts as an RyR1 luminal calcium sensor by

inhibiting release channel activity at normal SR calcium levels (~1 mM), and this

inhibition is relieved at high (> 4 mM) SR calcium levels.20,22 The influence of

calsequestrin on RyR activity appears to be transduced through a complex

conformational coupling mediated by junctin and triadin.22,102 Triadin and junctin

bind the RyR in a calcium-independent manner and prime the channel for activation.

At physiological levels of SR calcium, the binding of calsequestrin to the triadin-

junctin-RyR complex inhibits calcium release.22,102 However, interactions of

calsequestrin with triadin and junctin are calcium sensitive, such that extreme low (<

10 µM) and high (> 4 mM) levels of luminal calcium promote a conformational

change in the protein that weakens its interaction with triadin and junctin.266 Thus,

this relieves the inhibitory influence of the quaternary complex on calcium release.102

However, even small alterations in luminal SR calcium levels during release may
 19

result in subtle conformational changes in calsequestrin that are sufficient to influence

calcium release during EC coupling.21,102

Effects of SR calcium load on calcium release are also important during

normal activation of release by the voltage sensor. In fact, DHPRs can activate RyRs

over a broad range of SR calcium levels192 and thus fully deplete SR calcium,

resulting in a dramatic change in calcium release during EC coupling.137,192 The

ability of the voltage sensor to fully deplete the SR is likely mediated by long-range

allosteric effects by which luminal calcium levels alter inhibition of release channel

activity by cytosolic magnesium.144

RyR1 Regulators

Although calcium release during EC coupling is controlled by the DHPR, a

number of soluble molecules (e.g., Mg2+, ATP, calcium, and reactive oxygen species)

and associated proteins (e.g., FKBP12, calmodulin, homer, and PKA) also regulate

the activity of the SR calcium release channel. Cytoplasmic ATP activates whereas

cytoplasmic Mg2+ inhibits channel activity.62,63,217 Both the long and short form of

the homer protein activate calcium release through RyR1.251 Homer multimerizes at

its C-terminal coiled-coil and leucine zipper domains260 to form a junctional signaling

complex with RyR1.84,115,256 FK506-binding protein (FKBP12) binds

stoichiometrically to each protomer of the RyR1 tetramer.122 The binding of FKBP12

to RyR1 profoundly influences SR calcium release channel function by stabilization

of the fully open and closed states of the channel,122 controlling channel gating
 20

frequency,95 and modulating sensitivity to activation by calcium.169,170 Together, the

effects of FKBP12 on RyR1 function enhance the gain of skeletal muscle EC

coupling.14,16,228

Another important RyR1 regulatory protein is calmodulin (CaM), which also

binds stoichiometrically to each subunit of the tetramer.246 Calmodulin activates the

skeletal muscle calcium release channel at low (nanomolar) concentrations of calcium

and inhibits the channel at high calcium (millimolar) concentrations.39,239 However,

the precise role of the calmodulin interaction with RyR1 in modulating calcium

release during EC coupling is still unclear.182

Although specific mechanisms remain to be clarified, RyR1 activity is also

regulated by a variety of different post-translational modifications including oxidation

and phosphorylation. RyR1 possesses a number of highly reactive sulfhydryl

moieties that are susceptible to reversible S-nitrosylation, S-glutathionylation, and

disulfide oxidation.9,223,245 In particular, Aracena-Parks et al. found two regions of

the channel (residues 1-2401 and 3120-4475) that are major sites of S-nitrosylation

and S-glutathionylation.9 The combined response of these hyperreactive thiols

empowers the RyR1 tetramer to act as an SR redox sensor, resulting in regulation of

release channel activity in isolated systems by changes in the SR transmembrane

redox potential.83,109,110,165,183,264 However, redox modulation of SR calcium release

during EC coupling in intact muscle might be of little consequence because changes

in the SR transmembrane redox potential do not directly alter the amount of calcium

released from the SR during normal EC coupling.6,191

 21

RyR1 is also known to be phosphorylated by protein kinase A (PKA) and

calmodulin kinase II (CaMKII), the effects of which are still highly controversial.

PKA phosphorylation has been found to both activate,97,105 inactivate,247 or produce

no effect56 on RyR1 activity. In addition, PKA phosphorylation at Ser2843 has been

suggested to trigger FKBP12 dissociation from the release channel and subsequent

loss of FKBP12 regulation.199 However, other investigators have found that PKA

phosphorylation at Ser2843 does not alter RyR1 function or promote FKBP12

dissociation.219 Similarly, contradictory effects of CaMKII phosphorylation on RyR1

channel activity have also been reported.80,103

In addition, SR calcium release channel activity in skeletal muscle is

modulated by the direct action of a number of distinct pharmacological agents

including ryanodine, caffeine, 4-chloro-m-cresol, and ruthenium red. Ryanodine

binds with high affinity to the C-terminal pore-forming region of the channel.41

Ryanodine exerts a complex, dose-dependent effect on the activity of the release

channel. At low concentrations (~10 nM), ryanodine increases the frequency of

single channel openings in the absence of an effect on unitary conductance.38 At

intermediate concentrations (~1 µM), ryanodine locks the channel in a long-lived

open state with a decreased unitary conductance.38,202,218 High doses of ryanodine

(~100 µM) abolish ion conduction through the channel.269 Although not a

particularly effective calcium release trigger,139 caffeine is a low-affinity RyR agonist

that stimulates calcium release from the SR by binding to the release channel.215

Binding of caffeine to the release channel induces conformational changes in RyR1

 22

that result in increased release channel sensitivity to activation by other activators

such as calcium27,215 and voltage.18,255 4-Chloro-m-cresol, an agent originally used as

a preservative in commercial preparations of certain intravenous drugs, activates

calcium release through RyR1 and RyR2, but not RyR3, through interactions with the

C-terminus of the channel.85 In contrast, ruthenium red blocks SR calcium release49

by binding to the ion conduction pore of the channel.156 Given its highly cationic

nature, it is possible that, in addition to its effect of blocking the channel pore,

ruthenium red may also alter channel regulation through interactions with calcium

and magnesium regulatory sites. Thus, calcium release is affected by associated

proteins, soluble molecules, and pharmacological agents.

Calcium Influx

Control of extracellular calcium entry is less well-characterized. Store-

operated calcium entry (SOCE), which is activated by depletion of intracellular

stores, has been observed in skeletal muscle.137,142 Although calcium elevations

during single-twitch contractions are due exclusively to voltage-gated SR calcium

release,11,74 extracellular calcium influx through SOCE may be important during

prolonged tetanic stimulation and fatigue.157,189 SOCE in skeletal muscle requires

stromal interaction molecule 1 (Stim-1)153,220 and Orai1153 In addition, SOCE in

skeletal muscle is regulated by RyR1 as it is markedly reduced in myotubes derived

from RyR1/RyR3 double-knockout mice.189 Junctate may also modulate store-

operated calcium entry by forming a complex with the IP3 receptor and the transient
 23

receptor potential protein 3 (TRPC3).238 In addition, SOCE in skeletal muscle

requires normal triadic integrity because it is attenuated in myotubes in which

sarcolemma-SR junctions are disrupted.189

A second, novel calcium entry pathway activated by membrane depolarization

that requires conformational DHPR-RyR1 coupling was recently identified in skeletal

muscle (excitation coupled calcium entry, or ECCE).52 Although ECCE channels

exhibit a pharmacology that is strikingly similar to that of SOCE (blocked by Gd3+,

SKF-96365, and 2-APB), unlike conventional SOCEs, ECCE is activated by

prolonged or repetitive depolarization but not store depletion.52 Further, ECCE does

not require either Stim-1 or Orai-1, but does require both the voltage sensor (DHPR)

for activation and RyR1. It is clear, then, that SOCE and ECCE are controlled by

distinct molecular complexes.153 The dependence of both SOCE and ECCE on RyR1

activity raises the intriguing possibility that a conformational signaling interaction

between multiple triadic proteins simultaneously controls both calcium release during

EC coupling and activation of both capacitative and non-capacitative calcium entry

pathways.

I.1.3. Calcium Reuptake

Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA)

Clearance of calcium in skeletal muscle following release during EC coupling

primarily involves calcium reuptake into the SR by the sarco(endo)plasmic reticulum

Ca2+-ATPase (SERCA). Although calcium extrusion via plasma membrane calcium-

 24

ATPase (PMCA) pumps and Na+/Ca2+ exchangers balances calcium influx during EC

coupling in cardiac muscle, these calcium transport processes play only a minor role

in the clearance of myoplasmic calcium in skeletal muscle.8 SERCA-mediated SR

calcium reuptake controls the rate of muscle relaxation in skeletal muscle and is

responsible for maintaining a 10,000-fold calcium gradient across the SR membrane.

SERCA is a member of the family of P-type Ca2+-ATPases, named so because these

calcium transport proteins undergo autophosphorylation in which the γ-phosphate of

ATP is transferred to a highly conserved aspartyl residue in the cytoplasmic portion

of the protein.7 Thus, phosphorylation is an essential step in the mechanism of

calcium reuptake through SERCA.

There are five primary isoforms of SERCA encoded by three separate genes:

ATP2A1 (SERCA1), ATP2A2 (SERCA2), and ATP2A3 (SERCA3). SERCA1 is the

isoform found in fast-twitch skeletal muscle. The last two exons of ATP2A1 are

alternatively spliced to generate both adult (SERCA1a) and neonatal (SERCA1b)

skeletal muscle isoforms.30,267 Similarly, alternative splicing of the SERCA2 gene

also results in the production of two C-terminal splice variants (SERCA2a and

SERCA2b),155,265 as well as two relatively rare SERCA2 isoforms, SERCA2c98 and

SERCA2d.129 SERCA2a is the principal isoform present in cardiac, slow-twitch

skeletal, and neonatal skeletal muscle. SERCA2b is found in both nonmuscle tissues

and smooth muscle.257,259 SERCA3 is broadly distributed in platelets, lymphoid cells,

and some endothelial cells.258,259 Despite significant structural and primary sequence
 25

similarities, the tissue-specific expression patterns of each isoform suggests that the

different SERCA variants exhibit functionally distinct properties.24

The crystal structure of SERCA1a, a 994 amino acid (110 kDa) monomeric

protein located in the membrane of both terminal cisternae and longitudinal SR,160

has recently been solved.233,234 The single subunit contains 10 transmembrane

segments (M1-M10), with the bulk of the protein, including the N- and C-termini,

located in the cytoplasm (Figure I.1J). The structure can be divided into three parts: a

large cytoplasmic headpiece connected to an anchored SR transmembrane region via

a stalk domain.233 The cytoplasmic head is subdivided into three distinct domains: 1)

the actuator (A) domain (residues 1-50 and 131-238); 2) the nucleotide binding (N)

domain (residues 360-604); and 3) and the phosphorylation (P) domain (residues 330-

359 and 605-737) (Figure I.1J). The actuator domain facilitates engagement of the

gating mechanism and thereby regulates calcium binding and release.235 The γ-

phosphate of ATP is transferred to Asp351 in the P domain, whereas the adenosine

moiety binds to a site in the N domain.235 The luminal linkers between the

transmembrane segments are short except for the loop between M7 and M8. A

number of residues are essential for coordinating the cooperative binding of two

closely spaced calcium binding sites (5.7 Å apart) in the transmembrane region of the

protein. Site I is formed by Asn768, Glu771, Thr799, Asp800, Glu908, and the

oxygen atoms of two water molecules. Site II is formed by residues in M4 and M6

(Glu309, Asn 796, and Asp800) and three main-chain oxygen atoms from M4.167

Two calcium ions bind with high affinity to sites I and II on the cytoplasmic side of
 26

the protein at the beginning of the reaction cycle and are ultimately released from the

luminal side of the protein following ATP hydrolysis. For each ATP molecule

hydrolyzed per reaction cycle, two calcium ions from the cytoplasm are pumped into

the SR lumen in exchange for two or three H+ ions.263 In this way, SERCA1a utilizes

the energy stored in ATP to clear myoplasmic calcium during muscle relaxation.

Sarcolipin (SLN)

SERCA1 co-purifies with the low molecular weight regulatory protein,

sarcolipin (SLN).159 Sarcolipin (Figure I.1K) is a 31 amino acid protein with

sequence homology to the SERCA2-regulatory protein phospholamban (PLN).214

SLN structure includes a cytoplasmic N-terminus, a single transmembrane domain,

and a luminal C-terminus that contains an Arg-Ser-Tyr-Gln-Tyr (RSYQY) ER/SR

retention sequence.100 Similar to the effect of PLN on SERCA2 function, SLN is

thought to inhibit SERCA1 activity by decreasing its affinity for calcium and

lowering its Vmax.185 SERCA1 is also inhibited by NO, while its calcium affinity

decreases as pH is reduced.119 Additionally, SERCA1 function may also be regulated

by the luminal calcium binding protein, sarcalumenin (SAR; Figure I.1F).

Specifically, SAR interacts with SERCA1, and SR vesicles isolated from skeletal

muscle of SAR-deficient mice exhibit decreased calcium accumulation due to a

reduction in SERCA protein expression.262 However, because SERCA mRNA levels

in skeletal muscle are similar in wild-type and SAR-deficient mice, the interaction of

SAR with SERCA may serve to increase protein stability and reduce SERCA
 27

degradation. Thus, in the absence of SAR, a reduction in SERCA1 protein expression

results in a decrease in SR calcium reuptake and, subsequently, a slower time course

for relaxation.262

I.2. MITOCHONDRIA

Though SR calcium reuptake is the primary means of myoplasmic calcium

clearance during skeletal muscle relaxation, mitochondrial calcium uptake plays a

minor role.207 Furthermore, localization of mitochondria close to sites of calcium

release in striated muscle29,186,209,244 provides a physical basis for localized SR-

mitochondrial Ca2+ signaling and suggests that mitochondria participate in calcium

cycling at the triad.

I.2.1. Subcellular Localization in Muscle

Ogata and Yamasaki186 originally described a population of intermyofibrillar

I-band-limited mitochondria in electron microscopy studies of adult rat leg muscle.

All major muscle fiber types (red, white, and intermediate) exhibit pairs of slender

mitochondria encircling the myofibrils within the I-band on either side of the Z-

line.186 In addition, mitochondria are also found to cluster under the sarcolemma and

occasionally stack in longitudinal columns between the myofibrils in red muscle,

though columnar mitochondria are rarely observed in white muscle fibers.186

Quantitative analysis of nonfixed muscle fibers confirms the orientation of

mitochondria in transverse double rows with a sarcomeric periodicity of ~2 µm

 28

(Figure I.3A and 244), consistent with their close apposition to triads (Figure I.3B and

C). This ordered positioning results in a transverse lattice of mitochondria in skeletal

muscle fibers.244 A similar close CRU-mitochondrial juxtaposition is also observed

in rat myocardium,209,244 in which mitochondria occupy a much larger volume and

also run the length of the sarcomere, forming a longitudinal lattice between the

myofibrils.244

Not unlike the SR and mitochondria of adult striated muscle, domains of

endoplasmic reticulum (ER) are closely apposed to mitochondria in a variety of other

cell types, including smooth muscle cells, pancreatic acini, and hepatocytes.92 For

example, mitochondria associated with lamellae of rough ER can be recovered

following gentle subcellular fractionation procedures. This mitochondrial-associated

rough ER actually represents a morphologically and functionally distinct subset of

hepatic rough ER, and the isolated organelles retain a structural interaction similar to

that seen in intact hepatocytes.175 The ER-mitochondrial association in hepatocytes is

mediated by electron dense attachments or tethers (~10 nm to ~25 nm in length),

which are susceptible to disruption by trypsin-induced proteolysis.67 Remarkably, the

association of mitochondria with the ER could be strengthened by expression of a

short (5 nm) synthetic linker to enhance the connection between the two

compartments. Local calcium signaling between the two organelles was augmented

in accordance with the observed strengthening and reduction in ER-mitochondrial

spacing. As a result, ER-mitochondrial distance was found to be a critical

 29

determinant of local intercompartment calcium signaling, mitochondrial calcium

overload, and apoptotic cell death.67

Similarly, close association and tight coupling between mitochondria and the

CRU in muscle is likely to impact local intercompartment calcium signaling and

metabolic coupling during normal muscle activity, and also possibly influence

fatigability and muscle damage during eccentric exercise. Recently, a physical link

between the outer mitochondrial membrane (OMM) and the face of the junctional SR

opposite to that of the RyR1 feet has been suggested to anchor mitochondria to the

CRU.29 Such a linkage would not only preserve SR-mitochondrial colocalization

during muscle contraction and shortening, but also provide a structural basis for local

signaling between these two organelles during exercise, namely activity-dependent

ATP production.

I.2.2. Oxidative Phosphorylation (OXPHOS)

Sufficient ATP reserves are required to drive both actomyosin crossbridge

cycling and myoplasmic calcium removal during striated muscle contraction.

Following the power stroke, ATP binding to myosin causes dissociation of the

myosin head from actin, which is required for subsequent iterations of the crossbridge

cycle. In addition, ATP also provides the energy used to drive calcium reuptake by

the SERCA pumps during contractile relaxation. In fact, the majority of ATP

consumed during muscle contraction is used to fuel SERCA-mediated calcium

removal during contractile relaxation.2 In addition to ATP produced anaerobically

 30

during glycolysis and hydrolysis of phosphocreatine, aerobic metabolism within the

mitochondria represents a major source of cellular ATP production in muscle.

Anaerobic glucose metabolism in the myoplasm generates two molecules each

of ATP and pyruvate per glucose. Following glycolysis, the matrix enzyme complex

pyruvate dehydrogenase (PDH) catalyzes the decarboxylation of pyruvate, generating

acetyl-CoA that is then funneled into the tricarboxylic acid (TCA) cycle (Figure I.4).

The series of TCA cycle reactions produce reducing equivalents NADH and

FADH2.34 These products enter the electron transport chain (ETC) at the NADH-

dehydrogenase (complex I) and succinate dehydrogenase (complex II) complexes

located within the inner mitochondrial membrane (IMM). Transfer of electrons from

NADH to complex I results in accumulation of NAD+ which feeds back into the TCA

cycle for continued NADH generation. Complex I and II transfer the electrons from

NADH and FADH2, respectively, via Coenzyme Q (Q cycle), to complex III. It is

important to note that a small percentage of electrons flowing through the respiratory

chain can escape at complex I and the Q cycle. These free electrons combine with

molecular oxygen to produce superoxide anions which can then be converted to other

reactive oxygen species (ROS) (Figure I.4). In the final step, electrons are shuttled

from complex III to complex IV by cytochrome C with molecular oxygen serving as

the final electron acceptor.34 Electron transfer at complexes I, III, and IV is balanced

by proton pumping from the matrix to the intermitochondrial membrane space

(Figure I.4). This process generates an electromotive proton gradient used to drive

ATP synthesis from adenosine diphosphate (ADP) and inorganic phosphate (Pi) by
 31

the F1F0-ATPase (complex V).34 The sum of reactions from the beginning of the

ETC to ATP production by the F1F0-ATPase results is commonly referred to as

oxidative phosphorylation (OXPHOS).34 Theoretically, complete oxidation of one

glucose molecule (glycolysis and OXPHOS) yields 36-38 molecules of ATP.1

The relationship between fatigability and reliance on aerobic metabolism for

ATP generation between the different types of skeletal muscle underscores the critical

role of OXPHOS in muscle function during exercise or sustained muscle activity.

Specifically, slow-twitch muscle fibers are highly enriched in mitochondria, are more

dependent on OXPHOS for ATP production, and exhibit significant fatigue

resistance. On the other hand, fast-twitch glycolytic muscle fibers, with

comparatively fewer mitochondria, rely primarily on ATP generation from glycolysis

and hydrolysis of phosphocreatine. Since anaerobic ATP production is exhaustible

and generates ATP at a much lower rate than OXPHOS, fast-twitch glycolytic fibers

fatigue relatively quickly. Fast-twitch oxidative fibers, with a mitochondrial content

that is intermediate between slow-twitch and fast-twitch glycolytic fibers, are

relatively fatigue-resistant because they utilize a balance of aerobic and anaerobic

ATP generation.2 While the cellular mechanisms of skeletal muscle fatigue are

complex, a decline in cellular ATP levels during repetitive muscle activation clearly

contributes to the development of fatigue. For example, as the level of ATP

decreases during fatigue, the stimulatory effect of ATP on calcium release and

reuptake is lost,2 thus contributing to a reduction in muscle contraction. Therefore,

 32

limiting the development of muscle fatigue requires cellular mechanisms that

optimize ATP production with ATP utilization during muscle contraction.

Calcium Stimulation of OXPHOS

Cellular respiration and ATP production during skeletal muscle activity is

controlled by a number of mechanisms including [ATP]/[ADP], creatine kinase

activity, mitochondrial adenine nucleotide transport, and flux through the OXPHOS

machinery.17,34 Importantly, three of the mitochondrial dehydrogenases responsible for

generating NADH (pyruvate, NAD+-isocitrate, and α-ketoglutarate dehydrogenases)

during the TCA cycle are stimulated by elevations in matrix free calcium (Figure I.4).

First, the phosphatase that converts inactive pyruvate dehydrogenase phosphate to the

active pyruvate dehydrogenase is activated by calcium in the high nM to low µM

range. Importantly, the phosphatase requires Mg2+ and studies in permeabilized

mitochondria reveal that Ca2+ activates the phosphatase by decreasing its Km for

Mg2+.174 On the other hand, two other enzymes, NAD+-isocitrate dehydrogenase

(ICDH) and α-ketoglutarate dehydrogenase (α-KGDH), are activated directly by high

nM to low µM calcium.174 As reactions catalyzed by these enzymes may be rate-

limiting for flux through the TCA cycle, calcium stimulation of these enzymes

increases production of reducing equivalents used by the electron transport chain.

Finally, ATP generation via OXPHOS is further enhanced by calcium activation of

the ATP synthetic capacity of the F1F0-ATPase.229 In this way, mitochondrial calcium

uptake during EC coupling would serve to stimulate aerobic ATP production in order
 33

to help keep pace with increased ATP consumption associated with crossbridge

cycling and SERCA-mediated calcium sequestration during muscle activity (i.e.

excitation-metabolism coupling). However, while calcium-mediated enhancement of

mitochondrial ATP production has been documented in cultured cells and isolated

mitochondria, similar findings have yet to be demonstrated in adult skeletal muscle

fibers.

I.2.3. Mitochondrial Calcium Uptake

The concept of direct mitochondrial calcium uptake of calcium released

during muscle EC coupling has recently garnered significant support.92 Since

mitochondria colocalize to sites of calcium release, high calcium microdomains in

these discrete regions, coupled with the highly negative inner mitochondrial

membrane potential (-180 mV), provide a sufficient driving force for calcium uptake.

Calcium uptake occurs via the calcium uniporter (CaUP), a rapid calcium uptake

mode (RaM), and the mitochondrial ryanodine receptor (mRyR)(see Figure I.4 and
34
). In cardiac myocytes, rapid transient increases in mitochondrial calcium during

caffeine-induced SR calcium release result in significant mitochondrial calcium

uptake. This uptake is blocked by inhibition of the CaUP with ruthenium red, but not

by BAPTA, a rapid high-affinity calcium chelator.209 Similarly, Shkryl and

Shirokova demonstrated rapid “calcium tunneling” from the SR to the mitochondria

in slow- and fast-twitch skeletal muscle fibers during both electrical stimulation and

caffeine exposure.212 However, since mitochondria are associated with the SR on the
 34

side opposite to sites of RyR1-mediated calcium release (Figure I.3C), this most

likely reflects a local “through-space” coupling mechanism rather than a structural

tunneling between organelles. These data support the presence of a highly localized

or privileged calcium signaling communication between the SR and mitochondria in

striated muscle. Indeed, Rudolf et al.203 provided striking evidence for efficient

mitochondrial calcium uptake during physiological elevations in myoplasmic calcium

in intact skeletal muscle.203 Specifically, this study demonstrated in vivo

mitochondrial calcium uptake that occurs in synchrony with SR calcium release

during muscle contraction following motor nerve stimulation.203

In spite of the evidence discussed above, the relative importance of

mitochondrial calcium uptake in mammalian muscle during twitch and tetanic

stimulation remains controversial.2 For example, Lannergren et al.140 failed to detect

significant mitochondrial calcium uptake during SR calcium release in mouse fast-

twitch glycolytic fibers. In addition, even when mitochondrial calcium uptake during

tetanus and fatigue is detectable, its magnitude represents only a minor fraction of the

total calcium release, precluding a major role for mitochondria in shaping the global

myoplasmic calcium transient.2 Nevertheless, because of the relative paucity of

calcium buffers present in the mitochondrial matrix, even low levels of mitochondrial

calcium uptake may be sufficient to increase matrix calcium to levels required to

stimulate mitochondrial respiration and ATP production via excitation-metabolism

coupling.
 35

I.2.4. Mitochondrial Inhibition of Local Calcium Release

Additionally, SR-mitochondrial calcium signaling is not limited to excitation-

metabolism coupling. Local SR calcium release events, termed calcium sparks, were

first identified in cardiomyocytes, exhibit quantal spatiotemporal properties, and

represent elementary events of RyR-mediated calcium release. Specifically, calcium

sparks provide brief elevations in local calcium release that last on the order of a few

tens of milliseconds and exhibit a spatial width of ~1 µm.131 While calcium sparks

are not observed in mammalian skeletal muscle fibers under basal conditions, they are

unmasked following sarcolemmal permeabilization, disruption of mitochondrial

function, osmotic shock, and strenuous exercise.117,118,131,166,250,253

Several mechanisms have been proposed to explain the absence or

suppression of calcium sparks in intact adult mammalian skeletal muscle under basal

conditions. A degeneration of the T-tubular network in dedifferentiating cultured

adult skeletal muscle cells correlates with an increase in calcium spark frequency,131

consistent with a structural component to calcium spark suppression in adult muscle.

Similarly, calcium spark activity following strenuous exercise may involve a physical

disruption of the DHPR-RyR1 interaction.250 However, calcium sparks are also

readily observed in mechanically skinned fibers in which the DHPR-RyR1 interaction

is preserved.131 Therefore, mechanisms other than tight DHPR-RyR1 coupling likely

contribute to calcium spark suppression in skeletal muscle.

Subsequently, results from Shirokova and colleagues indicate a strong

contribution of functional mitochondria to calcium spark suppression in adult

 36

mammalian skeletal muscle.117,118,166 The potential role of strong mitochondrial

calcium buffering in spark suppression has been neither supported nor disproven.

Certainly, evidence presented in the previous section indicates that mitochondria are

capable of physiological calcium uptake. However, total mitochondrial calcium

buffering capacity is limited, and the degree to which mitochondria accumulate

calcium during a spark remains to be determined. Nonetheless, calcium spark activity

is markedly decreased by interventions that energize mitochondria and increased by

interventions that disrupt mitochondrial calcium uptake.117 For example, addition of

TCA cycle substrates diminishes calcium spark activity.117 Moreover, the onset of

calcium sparks in chemically permeabilized fibers occurs slowly in mitochondria-rich

slow-twitch oxidative muscle fibers, yet rapidly in mitochondria-poor fast glycolytic

fibers.118 In addition, spark activity in both fiber types is augmented by

mitochondrial calcium uptake blockers, protonophores that dissipate the

mitochondrial membrane potential, and antimycin A-mediated complex III

inhibition.117,118 Furthermore, oxidative insult produced by H2O2 application

accelerates the onset and increases the frequency of calcium sparks,118 whereas ROS

scavengers delay and/or suppress calcium spark activity.118,166 Clearly, mitochondrial

calcium uptake and a proper balance of ROS generation/scavenging are critical

determinants of calcium spark suppression in both skeletal117,118,166 and cardiac125

muscle.

How does mitochondrial content/activity inhibit local SR calcium release?

Since oxidation/nitrosylation of RyR1 destabilizes channel interactions with

 37

accessory proteins (e.g. calmodulin and FKBP12) and increases calcium activation

and the probability of RyR1 channel opening,81,111 calcium sparks in muscle may be

unmasked under conditions that increase the oxidative environment around the

CRU.118,166 Mitochondria are both intimately associated with the CRU (Figure I.3)

and provide a primary means of cellular ROS production and

scavenging/detoxification (Figure I.4). Specifically, while mitochondria produce

superoxide ions at complex I and during the Q cycle, this ROS production is balanced

by robust ROS scavenging (superoxide dismutase and catalase) mechanisms, as well

as glutathione- and thioredoxin-mediated ROS detoxification. Consequently,

mitochondria play a critical role in maintaining proper balance of the cellular redox

potential. Also, given their localization at the triad (Figure I.3), mitochondria may

influence calcium spark activity by regulating the local redox environment of the

CRU. Accordingly, under basal conditions, mitochondria inhibit local calcium

release by ensuring that the local redox environment around the CRU is sufficiently

reduced. On the other hand, calcium spark activity is increased under conditions that

overwhelm this local redox control mechanism and promote RyR1 oxidation either

directly (e.g. H2O2 application) or indirectly (low mitochondrial content, addition of

mitochondrial uncouplers, inhibition of mitochondrial calcium uptake.117,118,166

Accordingly, calcium spark activation following prolonged or fatiguing muscle

activity250 could result from unabated oxidation of the local SR-mitochondrial

microenvironment. In fact, ROS-mediated dysfunction in SR calcium

release/reuptake is thought to contribute to contractile decline during fatigue,

 38

suggesting that antioxidants may correct defects in local calcium release and increase

fatigue resistance.2

I.3. MUSCLE DYSFUNCTION AND DISEASE

The findings presented clearly indicate that proper calcium storage, release,

and reuptake are essential for normal physiological functioning of muscle excitation,

contraction, and relaxation. This conclusion is made all the more evident by the fact

that acute disruption in calcium homeostasis contributes to muscle fatigue and

damage following vigorous activity. Moreover, several human skeletal muscle

disorders (Malignant Hyperthermia, Central Core Disease, and Brody Disease) result

from dysfunction in the control and coordination of different aspects of these three

critical processes.

I.3.1. Fatigue

Muscle fatigue is defined as a reversible decline in performance (i.e. force

generation) with muscle activity. Several major mechanisms that cause fatigue have

been identified, most of which originate in the muscle itself and are a direct result of

increased ATP consumption during muscle activity.2 Muscle crossbridge cycling

during contraction and SERCA-mediated calcium reuptake during muscle relaxation

are energetically expensive processes. In fact, the energy demand of contraction can

be greater than one order of magnitude higher than resting demand.224 To

accommodate these sudden changes, muscle utilizes creatine phosphate (CrP). CrP is

generated from creatine and MgATP in a reaction catalyzed by creatine kinase. This
 39

reaction can be thought of as a “metabolic capacitance”, and is especially important in

fast-twitch glycolytic muscle.224 CrP, coupled with rapid activation of glycolysis

with the onset of activity, initially maintain a stable level of ATP. However, CrP

cannot supplement ATP production indefinitely because ATP itself is necessary to

generate CrP. Therefore, cellular ATP levels will eventually fall with continued

muscle activity. Furthermore, lactate and H+ are products of anaerobic glycolysis,

and their accumulation leads to muscle acidosis, which is classically associated with

fatigue. At the same time, ATP hydrolysis results in an increase in the concentration

of Pi and ADP, as well as Mg2+.2 More importantly, these metabolic changes during

repeated muscle activity underlie many of the alterations in calcium handling

associated with fatigue. RyR1 is inhibited by decreases in ATP and increases in

Mg2+. Similarly, calcium reuptake is inhibited by decreases in ATP and increases in

Pi.2

It is also worth noting that free radical and ROS/RNS production are

significant during muscle contraction.2,70,121,171,172 Mitochondrial OXPHOS and

membrane-bound NAD(P)H oxidoreductases (NOX) are significant sources of

superoxide in muscle.2 Superoxide can then react with NO· formed by nNOS

(neuronal nitric oxide synthase), to generate peroxynitrite which is a powerful

oxidant.33 Superoxide is readily scavenged in mitochondria by MnSOD (Manganese

superoxide dismutase) which generates H2O2, a freely diffusible cellular oxidant.34

The contribution of elevated levels of free radicals and ROS/RNS to muscle fatigue
 40

involve direct effects on RyR1-mediated SR calcium release and inhibition of SR

calcium reuptake.2

However, fatigue can arise from a breakdown at any point of the EC coupling

process, not only calcium release and reuptake. For example, changes in membrane

excitability due to alterations in Na+, K+, and/or Cl- fluxes, and subsequent action

potential failure, can result in fatigue. In addition, a decline in performance can occur

at the level of muscle contraction. Specifically, increasing levels of Pi and ROS, as

well as a drop in pH, decrease the calcium sensitivity of myofibrillar proteins,

reducing maximal Ca2+-activated force. As metabolites return to basal levels, fatigue

subsides and force generation recovers, typically within 1-2 hours.2

I.3.2. Exercise-Induced Muscle Damage

Unlike fatigue, muscle damage is long-lived and much slower to reverse,

appearing over a period of hours to days. Additionally, muscle injury can activate

satellite cells which are necessary to repair damage.2 Exercise-induced muscle

damage often occurs following lengthening (eccentric) contraction. Stretch of muscle

fibers as they contract can mechanically disrupt sarcomeres. As a consequence,

cytoskeletal and membrane structures become damaged. Activation of stretch-

activated cation channels, as well as loss of sarcolemmal integrity result in an

increase in myoplasmic calcium. Elevated calcium triggers proteolysis followed by

damage and initiation of muscle repair.196 Free radicals generated during contraction

can exacerbate membrane damage, especially mitochondrial lipid peroxidation.70 In

 41

short, eccentric contraction disrupts sarcomeric structure, causing a deficit in force

production.

An alternative explanation for the loss of muscle force induced by damage is

EC uncoupling,252 that is, the failure of an action potential to elicit sufficient SR

calcium release to trigger muscle contraction. Yet, EC uncoupling is likely secondary

to mechanical damage.196 In particular, failure of EC coupling following intense

exercise, especially eccentric contractions, is due in part to a disruption of triads. For

example, Takekura et al.226 found significant swelling of the SR and T-tubules,

increased appearance of longitudinal T-tubular elements, and a change in the normal

direction and nature of triads following downhill treadmill running. In addition, other

studies revealed a decrease in SR Ca2+ reuptake in skeletal muscle homogenates

following downhill exercise.50 This defect in SR Ca2+ reuptake following exercise-

induced muscle damage leads to a prolongation of contractile relaxation and a

decrease in SR Ca2+ load. Consequently, force production decreases due to the

reduction in SR Ca2+ available for release during successive contractions.40 Over the

long term, SR and T-tubular membrane damage and defective SR Ca2+ reuptake result

in an elevation of myoplasmic Ca2+. Indeed, peak muscle damage occurs 1-3 days

after strenuous exercise and most likely involves activation of Ca2+-dependent

proteases.50
 42

I.3.3. Malignant Hyperthermia

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal

muscle triggered by halogenated inhalation anesthetics (e.g., halothane) and

depolarizing muscle relaxants (e.g., succinylcholine).126,176 The hallmarks of MH are

increased muscle metabolism, muscle rigidity, and rapidly increasing body

temperature. Triggering agents initiate uncontrolled muscle contraction, resulting in

excessive ATP hydrolysis, acidosis, cyanosis, and heat generation. A hypermetabolic

state results as the muscle strives to replenish and maintain levels of ATP, ultimately

leading to breakdown of the muscle.8 In essence, an MH crisis arises from

uncontrolled skeletal muscle calcium release and contraction.

Several lines of evidence point to an abnormality in the SR calcium release

mechanism as the underlying defect in MH. First, in vitro contracture tests conducted

on muscle biopsies obtained from MH susceptible (MHS) patients have demonstrated

increased sensitivity to contractile activation in response to low concentrations of

caffeine and halothane.162 Second, genetic analyses of pigs susceptible to MH

identified a single point mutation in the RyR1 gene that results in the substitution of a

Cys residue for Arg615.94 This is the only mutation known to cause MH in pigs.161

Third, SR calcium release channels isolated from skeletal muscle of MHS porcine

muscle exhibited increased rates of calcium-induced calcium release; enhanced

sensitivity to activation by caffeine, halothane, and T-tubule depolarization; and

reduced inhibition by high concentrations of calcium and magnesium.76,176 Fourth,

three main clusters of mutations in RyR1 are linked to MHS in humans: mutations in
 43

the N-terminal (region 1: Cys35–Arg614), central (region 2: Asp2129 –Arg2458),

and C-terminal (region 3:Ile3916–Ala4942) regions of the protein. Finally, human

MH mutations in the N-terminal and central regions of the RyR1 protein are thought

to disrupt a critical interdomain interaction that normally acts to stabilize the resting

closed state of the release channel.116,133,261 As a result of this closed state

destabilization, MH mutations enhance release channel sensitivity to activation by a

wide range of RyR1 triggers, including caffeine, halothane, 4-chloro-m-cresol, and

membrane depolarization. Additionally, recent studies demonstrate that at least one

particular MH mutation, Tyr522Ser, increases RyR1 temperature sensitivity.48,81 In

particular, the mutation increases SR calcium leak, resulting in elevated resting

calcium levels, increased reactive nitrogen species (RNS) generation, and subsequent

S-nitrosylation of the mutant channel.81 S-nitrosylation renders the release channel

heat-sensitive. Thus, elevated temperature stimulates further calcium release, feeding

forward on the calcium-RNS-RyR1 modification cycle.81

Although linkage to the RyR1 gene is shown for more than 50% of all MH

kindreds, evidence also suggests that other MH-susceptible gene loci exist, including

chromosomes 17 (17q11.2-q24), 7 (7q21- q22), 3 (3q13.1), and 1 (1q31).173,177 Two

different point mutations of a highly conserved arginine residue in the III–IV linker of

the skeletal muscle DHPR α1s-subunit (R1086H and R1086C) have been confirmed

for the 1q31 locus.173,177 Similar to MH mutations in RyR1, the R1086H mutation in

the DHPR also increases the sensitivity of the SR calcium release mechanism to

activation by both caffeine and voltage.255 The finding that MH manifests from point
 44

mutations in both the skeletal muscle DHPR and RyR1, two key proteins of the EC

coupling machinery, reinforces the concept that MH arises from a dysfunction in

muscle EC coupling.

I.3.4. Central Core Disease

Central core disease (CCD) is a nonprogressive congenital myopathy, often

present at infancy, that is characterized by hypotonia, proximal muscle weakness, and

delayed attainment of motor milestones.213 CCD is diagnosed by the presence of

amorphous “cores” or regions in skeletal muscle that are devoid of mitochondria and

oxidative enzyme activity. In most cases, CCD has been linked to mutations in the

RyR1 gene, and these mutations are clustered in the same three regions of the RyR1

protein as those found for MH. Although certain mutations in RyR1 lead to the

coincidence of MH and CCD, some RyR1 mutations result in only increased MHS or

CCD in the apparent absence of MHS. RyR1 mutations in regions 1 and 2 that lead

to MH and CCD coincidence promote an excessive or uncompensated SR calcium

leak that results in a net depletion of SR calcium stores.12,178,232,236 However, the vast

majority of CCD mutations in RyR1 are found in the C-terminal part of the protein

(region 3).71,154,178 A subgroup of CCD mutations in region 3 occur within the

putative pore-lining region of the channel and appear to disrupt calcium activation79

and permeation through the channel. Because CCD mutations in the pore region of

the channel reduce calcium release during EC coupling in the absence of an effect on

SR calcium content, these mutations are suggested to lead to EC uncoupling.12,13 The

 45

effects of MH and CCD mutations in RyR1 on resting calcium, store content, and

calcium release during EC coupling are controversial. In general, mutations that lead

to an MH-selective phenotype result in “hypersensitive” release channels that operate

normally during EC coupling. However, muscle weakness in CCD arises from

mutations in RyR1 that lead to a reduction in calcium release during EC coupling.

MH and CCD coincidence arises from RyR1 mutations that cause uncompensated SR

calcium leak, which leads to a reduction in SR calcium content.77 Consequently,

mutations that result in MH and CCD coincidence result in SR calcium depletion,

thus reducing calcium release during EC coupling by altering both the amount of

calcium available for release and the effect of luminal calcium on the release

mechanism. In addition, for RyR1 mutations that lead to both MH alone and

MH/CCD coincidence, release channels exhibit increased sensitivity to activation by

a wide range of RyR1 activators (e.g., caffeine, halothane, 4-chloro-m-cresol,

voltage), and thus, are also susceptible to uncontrolled calcium release upon exposure

to volatile anesthetics. By contrast, mutations within the pore region of RyR1 that

result in CCD in the absence of increased MHS do not increase release channel

sensitivity to activation by RyR1 triggers or lead to SR calcium depletion.12,13,15

Rather, these mutations alter calcium gating/permeation in a manner that results in a

reduction in the ability of membrane depolarization to induce calcium release from a

full SR calcium store.77

 46

I.3.5. Brody Disease

Brody disease, first described in the literature in 1969 by Dr. Irwin A. Brody,

is a rare autosomal recessive muscle disease characterized by painless muscle

cramping and exercise-induced impairment of muscle relaxation.32 Muscles of the

legs, arms, and eyelids are typically affected, and episodes are exacerbated in the

cold. The selective defect of a “relaxing factor”32 was later identified as a mutation in

the ATP2A1 gene, which encodes the skeletal muscle SERCA isoform, SERCA1.184

However, similar to other muscle diseases, Brody disease is genetically

heterogeneous. In some families with Brody myopathy, no mutations in ATP2A1

have been identified. It was initially believed that defects in sarcolipin regulation of

SERCA1 might contribute to Brody disease in cases in which mutations in SERCA1a

could not be detected. However, no sarcolipin mutations were identified in 13

separate Brody families.163 Both point mutations and premature stop codons in

ATP2A1 have been identified in Brody disease families with autosomal-recessive

inheritance. In most cases, the mutations result in deleted functional domains that

lead to a reduction in SERCA1 function.164 Early studies showed that loss of

SERCA1 was restricted to fast-twitch skeletal muscle fibers. Yet, no difference in

total SERCA or SERCA1 protein levels was found between Brody disease and

control muscle homogenates.69,127 Nevertheless, a 50% reduction in calcium-

stimulated ATPase activity and a significant increase in the time required for return to

basal calcium levels following depolarization-induced calcium release was observed

in skeletal muscle of patients with Brody disease.23 Thus, SERCA1 defects result in
 47

the inability of SERCA activity to keep pace with calcium release during repetitive

stimulation. Consequently, myoplasmic calcium accumulates during vigorous

exercise, ultimately resulting in the delayed relaxation and muscle cramping

experienced by patients with Brody disease. Because muscle relaxation is not

completely impaired, only slowed, compensatory mechanisms ultimately work to

clear myoplasmic calcium over time. These include residual SERCA1/SERCA2

activity, calcium extrusion via PMCA pumps and Na+/Ca2+ exchangers, as well as

mitochondrial calcium uptake. Thus, the development of novel therapeutic strategies

designed to enhance calcium clearance and subsequent muscle relaxation by

augmenting PMCA, Na+/Ca2+ exchanger, and/or SERCA2 activity/expression might

prove useful for symptomatic treatment of patients with Brody disease.164

 48

I.4. OVERVIEW

Analogous to how an engine governor is designed to maintain a specified

speed, the SR functions to maintain vastly different resting myoplasmic and SR

calcium levels. This control involves a complex and highly regulated balance of

calcium storage, release, and reuptake coordinated by the activity of and interactions

between a diverse set of SR calcium regulatory proteins (Figure I.2). Under basal

conditions, SERCA pump activity and calcium binding to luminal SR proteins

(calsequestrin, HRC, junctate, and SAR) set resting myoplasmic calcium levels (~50-

100 nM) well below that required for myofilament activation and also establish a

10,000-fold calcium gradient across the SR membrane. During muscle excitation, the

control of low myoplasmic calcium is temporarily lost as membrane depolarization

stimulates rapid and dynamic release of calcium stores through activation of RyR1

release channels located in the terminal cisternae of the SR. As local calcium levels

decrease within regions of release, changes in luminal calcium act to self-limit further

release through a complex quaternary interaction between calsequestrin, triadin,

junctin, and RyR1. In addition, release is further limited by calcium-mediated

inhibition of RyR1 activity by high calcium microdomains formed on the myoplasmic

side of the release channel. Release is ultimately terminated by release channel

closure following membrane repolarization. Subsequent muscle relaxation and

restoration of low resting calcium levels are then achieved primarily through ATP-

driven calcium reuptake into the SR via SERCA1 activity. In this way, the SR in

skeletal muscle functions as a dynamic calcium governor that maintains low

 49

myoplasmic calcium levels during periods of muscle inactivity, establishes a large

calcium gradient across the SR that is used to drive calcium release and muscle

contraction during excitation, and provides for efficient SR calcium reuptake and

contractile decline during muscle relaxation. Even seemingly minor abnormalities in

the calcium storage, release, or reuptake functions of the SR calcium governor can

result in a profound impact on calcium cycling and, ultimately, lead to muscle

dysfunction and disease.

It is clear, then, the SR is the primary regulator of skeletal muscle calcium

cycling during EC coupling. Yet, the precise positioning of mitochondria adjacent to

CRUs in adult mammalian skeletal muscle situates these organelles to interact with

the SR and affect local calcium signaling at the triad. Specifically, I hypothesize that

the crosstalk between SR and mitochondria takes the form of a symbiotic

bidirectional communication analogous to the bidirectional coupling between RyR1

and DHPR (Figure I.5). Orthograde SR-mitochondrial signaling involves

mitochondrial calcium uptake following SR calcium release during EC coupling that

could subsequently stimulate mitochondrial respiration and ATP production to help

meet increased energetic demand during muscle activity (excitation-metabolism

coupling). On the other hand, functionally intact mitochondria inhibit undesired

localized SR calcium release likely by controlling the local redox environment of the

CRU. Thus, bidirectional SR-mitochondrial communication could provide a

powerful local control mechanism for integrating calcium release/reuptake and ATP

utilization during muscle contraction with ATP production and skeletal muscle
 50

bioenergetics. Potentially, the efficiency of this local control mechanism would rely

on precise colocalization of CRUs and mitochondria.

However, little is known about the genesis of the CRU-mitochondrial

coupling and, more specifically, mitochondrial disposition during skeletal muscle

development. Given the proximity of mitochondria to CRUs in adult muscle, it is

reasonable to hypothesize that mitochondria follow the developmental progression of

SR-T-tubule formation from a relatively random arrangement toward precise

organization. Therefore, the first aim of this research was to establish mitochondrial

localization in flexor digitorum brevis (FDB) fibers from young (2-4 weeks) and adult (2-

4 months) mice using confocal microscopy. The expectation was that mitochondria

would follow a similar developmental progression as the sarcotubular network, from

a relatively random arrangement with respect to the myofibril bundles in young

muscle toward a highly ordered localization in adult muscle. Accordingly,

mitochondrial-SR communication would mature during postnatal development.

Specifically, optimal orthograde signaling, and thus, the greatest

mitochondrial calcium uptake should occur in adult muscle, with fully matured CRUs

intimately associated with mitochondria. In support of this, the studies cited earlier

demonstrate calcium uptake in adult skeletal muscle mitochondria in vitro,207 in

situ,36,37,140,141 and in vivo,203 even if the physiological significance is disputed.

Conversely, calcium uptake should be minimal in mitochondria not directly apposed

to triads. Yet, published literature does not address how mitochondrial calcium

uptake varies with mitochondrial-SR colocalization. For this reason, the second aim
 51

of the current study was to characterize mitochondrial calcium uptake following

tetanic stimulation in FDB fibers throughout skeletal muscle development. In

keeping with the model, I anticipated an increase in mitochondrial calcium uptake as

mitochondria progressively target to CRUs.

A similar structural prerequisite of SR-mitochondrial colocalization would be

necessary for maximal calcium spark suppression, as well. In order to test this

assumption, the last aim of this work utilized an osmotic shock paradigm to induce

calcium sparks in intact young and adult FDB fibers. CRU-targeted mitochondria

should buffer osmotic shock-induced sparks, just as energized and metabolically

active mitochondria inhibit localized calcium release in permeabilized muscle

fibers.117,118 Therefore, I expected that Ca2+ spark suppression to increase during

postnatal development in parallel with an increase in mitochondrion-CRU targeting.

In conclusion, my initial characterization of mitochondrion-CRU targeting

throughout skeletal muscle development provided the foundation for investigating

both bidirectional calcium and ROS signaling between SR and mitochondria.

Furthermore, showing that CRU-localized mitochondria take up calcium and buffer

sparks more efficiently than those not localized to CRUs would support a privileged

interaction between SR and mitochondria and argue for a significant role of

mitochondria in skeletal muscle calcium cycling.

 52

Figure I.1. Structural Features of Key Calcium Regulatory Proteins of the Skeletal

Muscle Sarcoplasmic Reticulum

A) Calsequestrin. B) Triadin. C) Junctin. D) Histidine-rich calcium binding protein

(HRC). E) Junctate. F) Sarcalumenin. G) Ryanodine receptor (RyR1). H) Junctophilin

type 1 (JP-1). I) Junctophilin Protein-45 (JP-45). J) SR/ER Ca2+-ATPase (SERCA).

K) Sarcolipin.
 53

Figure I.2. Location and Interactions of the Different SR Calcium Regulatory

Proteins Within One Side of the Triad

Calsequestrin, triadin, junctin, junctate, RyR1, JP-1, JP-45, SERCA, and sarcolipin

are depicted using the schematics shown in Figure I.1. For simplicity, sarcalumenin

and HRC proteins are shown as gray four-point stars and white triangles, respectively,

and SERCA is shown only in the longitudinal SR. Potential interactions between

RyR1 and the α1s- and β1a-subunits of the skeletal muscle DHPR, and the C-terminus

of the α1s-subunit and JP-45 are also shown.

 54

Figure I.3. Mitochondria are Precisely Localized Adjacent to the Calcium Release

Unit in Adult Mammalian Fast-Twitch Skeletal Muscle

A) Confocal image of a single flexor digitorum brevis (FDB) skeletal muscle fiber

obtained from an adult mouse (4 months of age) stained with the mitochondrial-selective

dye tetramethylrhodamine ethyl ester (TMRE). TMRE fluorescence along the line of

interest marked in A shows characteristic doublets of fluorescence with a sarcomeric

periodicity of ~2 µm (inset). B and C) Representative low (B) and high (C) resolution

electron micrographs of FDB muscle fibers obtained from an adult mouse (4 months of

age). Mitochondria (open arrows) are aligned adjacent to the triad (arrowheads), on

either size of the Z-line (solid arrows).

 55

Figure I.4. Schematic Representation of Interactions Between Components of the

Mitochondrial Calcium Transport Systems, the Tricarboxylic Acid (TCA) Cycle,

and the Electron Transport Chain (ETC)

Elevations of calcium in the mitochondrial matrix stimulate pyruvate dehydrogenase

(PDH), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), and

the ATP synthetic activity of the F1F0-ATPase (complex V). Low levels of superoxide

anions (reactive oxygen species, ROS) are generated as byproducts of electrons being

passed to O2 at complex I and during the Q cycle (Q). OAA, oxaloacetate; Ac-CoA,

acetyl coenzyme A; CS, citrate synthase; Acon, aconitase; α-KG, α-ketoglutarate; MDH,

malate dehydrogenase; I–V, complexes I–V; C, cytochrome c; OMM, outer

mitochondrial membrane; IMM, inner mitochondrial membrane; NHE, sodium-hydrogen

exchanger; NCX, sodium-calcium exchanger; CaUP, calcium uniporter; RaM; rapid

mode calcium transporter; mRyR, mitochondrial ryanodine receptor.

 56

Figure I.5. Bidirectional SR-Mitochondrial Signaling in Skeletal Muscle

Orthograde SR-mitochondrial signaling (solid lines) involves calcium release during

excitation-contraction (EC) coupling being taken up into adjacent mitochondria to

stimulate oxidative phosphorylation (OXPHOS) and ATP production. Retrograde

mitochondrial-SR signaling (broken lines) involves the influence of mitochondrial

reactive oxygen species (ROS) production and scavenging/detoxification on the local

redox environment and local Ca2+ spark activity of the Ca2+ release unit (CRU). SR,

sarcoplasmic reticulum; RyR1, type 1 ryanodine receptor; DHPR, dihydropyridine

receptor; TT, transverse tubule; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase.

 57

CHAPTER 1: MITOCHONDRIAL SUBCELLULAR LOCALIZATION

 58

1.1. RATIONALE

A striking characteristic of adult mammalian skeletal muscle is the precise

positioning of mitochondria adjacent to the triad or calcium release units (CRUs).

This observation has prompted investigation of mitochondrial calcium uptake during

excitation contraction (EC) coupling and the subsequent effects on the physiology of

muscle contraction and relaxation.36,140,141,203,207,212 Yet, prior studies have not

determined the origins and maturation of this interorganelle coupling, which is the

foundation for any proposed functional interaction between the two. The goal of the

following research was to fill in this gap of knowledge by characterizing

mitochondrial localization in fast-twitch skeletal muscle throughout postnatal skeletal

muscle development. I hypothesized that, analogous to the development of the SR

and T-tubule components of the CRU, mitochondria would lack the proper

disposition in young fibers but would gradually progress towards the precise

positioning typical of adult skeletal muscle during postnatal development.

1.2. METHODS

1.2.1. Preparation of Muscle Fibers

Mice were housed at the University of Rochester School of Medicine and

Dentristry (URSMD) and sacrificed immediately prior to experiments by CO2

overdose by regulated CO2 delivery followed by cervical dislocation, in accordance

with protocols approved by the University Committee on Animal Resources at

URSMD. Single acutely dissociated flexor digitorum brevis (FDB) muscle fibers
 59

were isolated from young (0.5 and 1 month old) and adult (2 and 4 month old)

C57/Bl6 mice as described previously.152 Briefly, FDB muscles were dissected from

mouse hind limbs in Ringer’s solution (in mM: 146 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2,

10 HEPES; pH 7.4). Single intact muscle fibers were obtained by enzymatic

dissociation with 1 mg/ml Collagenase A dissolved in Ringer’s solution and gentle

shaking in a water bath for 45 minutes at 37°C. Single FDB fibers were then

liberated by trituration with glass pipettes of decreasing bore size. Fibers were plated

on glass coverslips and allowed to settle for 1-2 hours before dye loading.

1.2.2. Confocal Imaging and Analysis of Mitochondrial Localization and

Membrane Potential

To assess mitochondrial position with respect to T-tubule localization, FDB

fibers obtained from 0.5 and 4 month-old mice were simultaneously loaded with 100

nM MitoTracker® Green FM (MTG, mitochondria marker) and 5 µM di-8-ANEPPS

(sarcolemmal potentiometric dye). Di-8-ANEPPS and MTG fluorophores were

sequentially excited using 543 nm (8X attenuation, 605/75 nm emission) and 488 nm

(4X attenuation, 515/30 nm emission) lasers, respectively. All images (512x512, 0.09

µm/pixel) were acquired and averaged (n = 4) using a Nikon Eclipse C1 Plus

Confocal microscope (30 µm pinhole) equipped with a SuperFluor 40X 1.3 NA oil

objective.

To assess relative changes in mitochondrial membrane potential during

development, FDB fibers obtained from 0.5, 1, 2, and 4 month-old mice were loaded
 60

with 1.5 µM JC-1 in Ringer’s solution for 20 minutes at 37°C. With the focal plane

positioned at the center of the fiber, JC-1 was excited using a 488 nm laser (8X

attenuation, 515/30 nm and 605/75 nm emission) to produce an average 47.7 µm x

47.7 µm image (n = 4). A composite average ratio (red/green) image of JC-1

aggregate (red) and JC-1 monomer (green) fluorescence was subsequently created

offline. A 20 µm line along the longitudinal axis of the fiber was drawn to generate a

mean XY ratio profile with X values representing fiber width and Y values

representing the longitudinally averaged fluorescence ratio. Because each line length

varied with fiber width, all X values were normalized to the longest X value, yielding

a percent of the fiber width for each profile. The ratio values were binned and

averaged every 2% according to their corresponding X-coordinates. To enable

comparison between different fibers, images were recorded using identical laser

power, photomultiplier sensitivity, and were processed using identical values for

contrast and brightness. Images were processed and analyzed using NIH ImageJ and

AutoQuant® AutoDeblur® & AutoVisualize software packages. A 2D blind

deconvolution was applied to all images for display purposes. Statistical significance

determined with Kruskal-Wallis one-way ANOVA on ranks with Dunn’s post test.

Results were verified in a separate set of imaging studies by quantifying the

ratio of tetramethylrhodamine ethyl ester (TMRE,10 nM) fluorescence, which is ∆ψm-

dependent, to MTG (300 nM) fluorescence, which accumulates in mitochondria

regardless of ∆ψm.59 TMRE and MTG were sequentially excited using 543 nm (8X
 61

attenuation, 605/75 nm emission) and 488 nm (64X attenuation, 515/30 nm emission)

lasers, respectively. Images were acquired and analyzed as described above.

1.2.3. Electron Microscopy (EM)

FDB muscles were fixed in situ with fixative solution (3.5% glutaraldehyde in

0.1 M NaCaCo buffer, pH 7.4) at room temperature. Small bundles of fixed muscle

fibers/cells from FDB muscles were then post-fixed in 2% osmium tetroxide (OsO4)

in the same buffer for 2h and block-stained in aqueous saturated uranyl acetate.

Specimens were then dehydrated in graded ethanol and acetone, infiltrated with Epon

812-acetone (1:1) mixture for 2 h, and then embedded in Epon. Ultrathin sections

(approximately 30-40 nm) and semi-thin sections (100 nm) were cut in Leica Ultracut

R (Leica Microsystem, Austria) using a Diatome diamond knife (Diatome Ltd. CH-

2501 Biel, Switzerland) and stained in 4% uranyl acetate and lead citrate. All

sections were examined with a FP 505 Morgagni Series 268D electron microscope

(Philips) at 60kV equipped with Megaview III digital camera and Soft Imaging

System (Germany).29

CRU and mitochondrial density (and their position relative to the sarcomeres)

was determined from 5 to 8 micrographs (at 14000 X) of non-overlapping regions

that were randomly collected from longitudinal sections of internal fiber areas. In

each EM image, the number of triads and mitochondria was determined, as well as

the incidence of mitochondrial positioning with respect to the I- and A-bands. If an

individual mitochondrion extended from one band to the other, it was counted in
 62

both. CRUs and mitochondria were marked and counted in each micrograph and the

area of the image was determined.29

1.3. RESULTS

1.3.1. Mitochondria-CRU Colocalization Increases During Postnatal Development

Using confocal microscopy, I determined mitochondrial subcellular

localization in single isolated FDB muscle fibers at early and late stages of postnatal

development (0.5 and 4 months). In each case, fibers were co-loaded with di-8-

ANEPPS to stain surface and T-tubule membranes and MTG, a mitochondria-

selective fluorescent probe. Mitochondria in FDB fibers from 0.5 month-old mice are

arranged primarily in subsarcolemmal and longitudinal clusters (Figure 1.1A and B,

green signal). On the other hand, a primitive T-tubule system has begun to develop

even in young 0.5 month-old fibers, as evidenced by fibers exhibiting double rows of

transverse di-8-ANEPPS staining, consistent with the known location of the T-tubule

network at the A-I junction in mammalian muscle (Figure 1.1A and B, red signal).

Strong colocalization of MTG and di-8-ANEPPS signals along the sarcolemma

(Figure 1.1A and B, yellow areas) is indicative of a subsarcolemmal population of

mitochondria in 0.5 month old FDB fibers. The absence of significant transverse

MTG and di-8-ANEPPS colocalization suggests that mitochondria are not well-

localized to the triad junction at this stage of postnatal development. Indeed, electron

microscopy (EM) analysis reveals only a small fraction of I-band-limited

 63

mitochondria adjacent to triads. At 0.5 months of age, the majority of mitochondria

are clustered beneath the sarcolemma and within the myofibrillar space (Figure 1.2A)

Subsarcolemmal and longitudinal clusters of mitochondrial fluorescence are

not typically observed in FDB fibers from adult (4 months-old) mice (Figure 1.1C

and D, Figure 1.2B). Instead, mitochondria create a strong pattern of MTG

fluorescence between double rows of transverse di-8-ANEPPS fluorescence. This

pattern of alternating rows of MTG and di-8-ANEPPS fluorescence with a periodicity

of ~2 µM is consistent with the location of mitochondria on either side of the Z-line

in adult muscle (Figure 1.1B and E, Figure 1.2B).

1.3.2. JC-1 Emission Distribution and Magnitude are Altered During Postnatal

Development

Progressive positioning of mitochondria to the I-band throughout postnatal

skeletal muscle development is also observed in FDB fibers stained with the

mitochondrial membrane potential indicator, JC-1 (Figure 1.3A). Since JC-1 is a

potentiometric mitochondrial probe, it enables a relative comparison of mitochondrial

membrane potential across each of the developmental time points studied, in addition

to its use as an independent confirmation of mitochondrial localization. Specifically,

monomers of JC-1 emit green fluorescence and predominate in less hyperpolarized

mitochondria, while JC-1 aggregates emit red fluorescence and accumulates in

mitochondria exhibiting a very negative membrane potential. In both young and

adult muscle fibers, puncta of red JC-1 fluorescence were clearly visible, though
 64

young fibers exhibited more puncta than adult fibers, and red JC-1 fluorescence was

typically more concentrated adjacent to the sarcolemma (Figure 1.3A, left). We also

found that the average ratio of JC-1 aggregate (red) to JC-1 monomer (green)

fluorescence (Figure 1.3A, right) was significantly (p < 0.001) greater in FDB fibers

isolated from 0.5 and 1 month-old mice (1.21 ± 0.02 and 1.58 ± 0.01 at 50% fiber

width, respectively) compared to that observed for fibers from 2 and 4 month-old

mice (0.82 ± 0.01 and 0.74 ± 0.01 at 50% fiber width, respectively) (Figure 1.3B).

The decrease in JC-1 emission ratio throughout postnatal development likely reflects

a global change in mitochondrial polarization in addition to a decrease in the number

of hyperpolarized mitochondria. Additionally, independent measurements of

mitochondrial membrane potential with TMRE at the transitional 1- and 2-months

time points, revealed a similar change in mitochondrial polarization during

development (Figure 1.3C). The mean TMRE/MTG fluorescence ratio in 1 month-

old fibers (1.5 ± 0.03, at 50% fiber width) was significantly (p < 0.001) higher than in

2 month-old fibers (1.3 ± 0.02, at 50% fiber width).

1.4. DISCUSSION

To date, most skeletal muscle structural studies have focused on two major

areas: 1) muscle cytoarchitecture, including the filament systems (e.g. actin, myosin,

titin)43,57 and the cytoskeleton (e.g. dystrophin-glycoprotein complex);3,107 and 2) the

sarcotubular system.82,90 Certainly, the focus on the former is due to its role in

driving muscle contraction and maintaining sarcomeric integrity. The emphasis

 65

placed on the sarcotubular system is best described in a review published in the early

stages of understanding skeletal muscle differentiation and development.221 The

authors write, “A nerve impulse passing along the T-tubules affects some as yet

unknown change in the SR membranes (the T-tubule does not open directly into the

lateral cisternae) that causes these membranes to lose some of the Ca2+ they have

accumulated. This efflux of Ca2+ from SR membranes triggers contraction. It is

obvious, therefore, that both the T-system and the SR system of skeletal muscle cells

have vitally important physiological roles, and the sequence of development of these

structures is of considerable interest.”221 Simply stated, the sarcotubular system

mediates the process of EC coupling, warranting extensive characterization. The

current study can be explained in the same context. Mitochondria are postulated to

play a significant role in calcium cycling at the triad during skeletal EC coupling

which necessitates structural characterization of the mitochondrial-SR association.

The close juxtaposition of mitochondria and triads in adult striated muscle has

been described in several papers.186,187,198,244 Ogata and Yamasaki186,187 described

mitochondrial disposition in skeletal muscle in the greatest detail. All muscle types

(red, white, and intermediate) exhibit pairs of mitochondria on either side of the Z-

line encircling the myofibrils. Consistent with these original findings,186 I find close

juxtaposition of mitochondria and triads at the A-I band junction in FDB fibers

isolated from adult (2-4 months of age) mice, with limited numbers of mitochondria

clustered beneath the sarcolemma and between the myofibrils.

 66

However, the major contribution of this work is in the novel characterization

of mitochondrial disposition at early stages of postnatal fast-twitch skeletal muscle

development. To begin, 0.5 and 1 month were chosen for study as transitional time

points during which SR and T-tubule membranes develop and mature.82,90 Though

triads are present in some neonatal skeletal muscle,82 they are few in number and are

longitudinally oriented. During early postnatal development, orientation of the

sarcotubular network becomes more regular, resulting in an increase association

between the SR and T-tubules, and subsequently, an increase in triad formation.29,82,90

Similarly, mitochondria progress from a relatively random disposition toward precise

organization. In fibers isolated from 0.5 month-old mice, mitochondria are

predominantly clustered between myofibril bundles and beneath the surface

membrane. From 0.5 to 4 months, there is a gradual disappearance of the

longitudinal mitochondrial clusters as mitochondria relocalize to the I-band and

associate with CRUs. Moreover, the parallel maturation of CRUs and

mitochondrion-CRU association suggests a common driving mechanism.

We provided evidence for a possible mechanism for establishment and

maintenance of SR-mitochondria association during postnatal development and in

adult muscle.29 This study described electron-dense tethers of approximately 10 nm

that bridge mitochondria and parajunctional regions of SR terminal cisternae. Tethers

persist even when the junction between SR and mitochondrial membranes is

“stretched” by subjecting the muscle fibers to osmotic shock, indicating that the

structure is fairly robust. The postnatal increase in mitochondrial localization to the I-

 67

band, increase in mitochondria and CRU density, and increase in mitochondrion-CRU

pairing (Table 1.1), concurrent with an increase in tethers, suggests that tethers help

to establish mitochondrial-SR association. Furthermore, tethers may contribute to

anchoring of mitochondria adjacent to CRUs, which would be especially important

under the mechanical stress of muscle contraction. Interestingly, de Brito et al.

recently identified the protein which stabilizes and maintains close endoplasmic

reticulum (ER) and mitochondrial juxtaposition in mouse embryonic fibroblasts

(MEFs).72 Specifically, ER-mitochondrial tethers are formed as a complex of ER

membrane-bound mitofusin 2 (MFN2), a dynamin-related GTPase, with

mitochondrial MFN1 or MFN2. Loss of MFN2 induces mitochondrial fragmentation,

swelling/aggregation of the ER, and a decrease in ER-mitochondrial association.72

Thus, MFN2 is a strong candidate molecule for the skeletal muscle mitochondrial-SR

tethers. At the very least, the identification of a “tethering protein” will contribute

considerably to studying the mechanism of mitochondrial-CRU targeting.

Additionally, the cellular signaling cascades that trigger and orchestrate

mitochondrial organization throughout postnatal skeletal muscle development require

further study. Comparison of the developmental program of fast glycolytic muscle

(e.g. FDB) with fast oxidative (e.g. extensor digitorum longus, EDL) and slow-twitch

(e.g. soleus) muscle may reveal possible mechanisms. Given that close

mitochondrion-CRU apposition seems to be characteristic of all adult mammalian

striated muscle,186,187,198,244 it is likely that mitochondria in oxidative skeletal muscle

follow the same progression as in the FDB examined in this study. However,
 68

longitudinal columns of mitochondria are common in red muscle,186 which also has

the greatest mitochondrial density of all muscle types. Perhaps, differences in the

isoforms of contractile proteins expressed,58 muscle innervation,123 or global calcium

dynamics44 may elucidate what drives the dissociation of mitochondrial clusters in

glycolytic but not oxidative muscle.

Despite unresolved questions of the mechanism, the data presented here

clearly indicate that mitochondrial-triad targeting is a highly coordinated and

developmentally regulated process. Consequently, mitochondrial calcium uptake and

function during muscle excitation are likely to differ significantly during skeletal

muscle development. For example, I found that the relative mitochondrial membrane

potential is most negative at 1 month, a dynamic time during which mitochondrial

targeting to CRUs lags behind triad maturation. Thus, the degree of mitochondrial

calcium uptake, ATP production, and reactive oxygen species (ROS) generation

during muscle contraction are likely to depend on both development and maturation

of triads and the evolution of mitochondrial-SR coupling.

 69

Table 1.1. Quantitative Analysis of CRUs and Mitochondria

The density of SR/TT junctions (CRUs) and mitochondria was determined by

counting their number in 6 to 8 randomly collected micrographs from each specimen

with no overlapping regions (at 14,000 X), all from internal areas of the fibers.
 70

Figure 1.1. Mitochondria-CRU Colocalization Increases During Postnatal

Development

Confocal images of representative FDB fibers obtained from 0.5 (A and B) and 4 (C

and D) month-old mice stained for surface and T-tubule membranes (red, 10 µM di-

8-ANEPPS) and mitochondria (green, 100 nM MitoTracker® Green FM). B and D)

Higher magnification images of the boxed regions shown in panels A and C,

respectively. E) Schematic representation of sarcolemmal (red) and mitochondrial

(green) staining at 0.5 (left) and 4 months (right).

 71

Figure 1.2. Electron Microscopy (EM) Analysis of Increased Mitochondrion-CRU

Positioning

Representative electron micrographs (longitudinal sections) of 0.5 (A) and 4 (B)

month FDB fibers. EM labeling key: arrowhead, sarcolemma (SM); solid arrow, z-

line; longitudinal mitochondria, large open arrows; I-band-limited mitochondria,

small open arrows; open stars, locations between myofibrils missing mitochondria.
72
 73

Figure 1.3. Mitochondrial Distribution and Membrane Potential are Altered

During Postnatal Development

A) JC-1 confocal image overlay (J-aggregate [red] and JC-1 monomer [green]

fluorescence, left) and false-colour JC-1 ratio images (J-aggregate/JC-1 monomer,

right) in representative FDB fibers obtained from 0.5, 1, 2, and 4 month old mice. B)

Representative JC-1 ratio image (left) used to calculate mean (± SE) JC-1 ratio spatial

profiles (right) in FDB fibers obtained from 0.5 (filled circles), 1 (open circles), 2

(filled triangles), and 4 (open triangles) month-old mice. To determine the JC-1

profile for each ratio image, a 20 µm long line of interest was drawn across the

longitudinal axis of the fiber to generate a mean XY profile with X values

representing relative fiber width and Y values representing the longitudinally

averaged ratio. Data from each population was normalized, binned and averaged as

described in the Methods section. 1.21 ± 0.02, n = 109 (0.5 m); 1.58 ± 0.01, n = 43 (1

m); 0.82 ± 0.01, n = 49 (2 m); and 0.74 ± 0.01, n = 75 (4 m) at 50% fiber width. *p <

0.001 compared with 4 months at 50% fiber width. C) Mean TMRE/MitoTracker®

Green FM (MTG) ratio spatial profiles of FDB fibers obtained from 1 (open circles)

and 2 (filled triangles) month-old mice. 1.5 ± 0.03, n = 113 (1 m); and 1.3 ± 0.02, n =

78 (2 m) at 50% fiber width. Bars: 5 µm.

 74

CHAPTER 2: MITOCHONDRIAL CALCIUM UPTAKE

 75

2.1 RATIONALE

My characterization of close mitochondrion-CRU colocalization provides the

structural basis for orthograde SR-mitochondrial signalling (Chapter 1), namely

mitochondrial calcium uptake. Previous studies have demonstrated mitochondrial

calcium uptake in adult skeletal muscle during physiological elevations in

myoplasmic calcium.36,37,140,141,203,212 To demonstrate the privileged nature of the SR-

mitochondrial communication, these studies utilized pharmacological interventions to

block mitochondrial calcium uptake, disrupt mitochondrial function, and alter the

calcium microdomain.36,140,212 In contrast, the following research takes advantage of

developmental differences in mitochondrial positioning with respect to sites of

calcium release to test the intimacy of SR-mitochondrial orthograde signaling.

Specifically, I set out to determine how mitochondrial calcium uptake during EC

coupling varied with mitochondrial-CRU targeting in fast-twitch skeletal muscle

throughout postnatal development. The expectation was that the magnitude of

calcium uptake during EC coupling would be greatest in triadic mitochondria,

especially in adult muscle. On the other hand, longitudinal mitochondria, without

exclusive access to the calcium microdomain, would accumulate less calcium.

2.2. METHODS

2.2.1. Preparation of Muscle Fibers

Single acutely dissociated FDB muscle fibers were isolated from young (1

month old) and adult (4 month old) C57/Bl6 mice as described in Section 1.2.1.
 76

Fibers were plated in a modified Ringer’s solution (in mM: 140 NaCl, 4 KCl, 1

MgSO4, 1.8 CaCl2, 5 NaHCO3, 10 Glucose, 10 HEPES; pH 7.4) on glass bottom

tissue culture dishes and allowed to settle for 20 minutes before dye loading.

2.2.2. Measurements of Myoplasmic Calcium

Fibers were loaded with 4 µM Mag-Fluo-4/AM for 25 minutes at room

temperature, followed by 20 minutes in dye-free solution supplemented with 25 µM

N-benzyl-p-toluene sulfonamide (BTS, to block contractions53). A rectangular region

of Mag-Fluo-4 loaded fibers was excited at 488 nm. Fluorescence emission at 517

nm was collected at 10 kHz using a photomultiplier detection system. Data was

analyzed using Clampfit and SigmaPlot software packages.

2.2.3. Confocal Imaging and Analysis of Mitochondrial Calcium Uptake

Fibers were loaded first with 10 µM Rhod-2/AM for 30 minutes at room

temperature, then loaded with either 300 nM MitoTracker® Green (MTG) or 10 µM

Fluo-4/AM, followed by either 20 minutes in dye-free solution supplemented with 25

µM BTS or 30 minutes in dye-free solution plus 25 µM EGTA-AM. Additional

experiments were conducted in fibers loaded with 10 nM TMRE and 10 µM Fluo-

4/AM. Green (MTG, Fluo-4) and red (Rhod-2, TMRE) fluorophores were

sequentially excited using 488 nm (16X attenuation, 515/30 nm emission) and 543nm

(8X attenuation, 605/75 nm emission) lasers, respectively. Time series (11 frames,

256x256 pixels/frame, 0.195 µm/pixel) were acquired using a Nikon Eclipse C1 Plus
 77

Confocal microscope (30 µm pinhole) equipped with a SuperFluor 40X 1.3 NA oil

objective. Tetanic trains (500 ms at 2.5 s interval, start-to-start) of electrically-

evoked calcium release were elicited with high frequency (8V, 5 ms, 100 Hz)

stimulation using an extracellular agar-plug electrode placed near the fiber of interest.

To enable comparison between different fibers, images were recorded using identical

laser power, photomultiplier sensitivity, and were processed using identical values for

contrast and brightness. Image sequences were processed and analyzed offline using

NIH ImageJ and AutoQuant® AutoDeblur® & AutoVisualize software packages. A

2D blind deconvolution was applied to all images for display purposes.

To generate a mean fluorescence value for triadic mitochondria, a 2 µm line

along the longitudinal axis of the fiber was drawn to generate a mean XY ratio profile

with X values representing fiber length and Y values representing the longitudinally

averaged fluorescence ratio. Values for profile peaks (I-band fluorescence) and

troughs (A-band fluorescence) following stimulation were normalized to their

respective values at rest. It is worth noting that image acquisition was staggered with

the beginning of each tetanus to minimize movement artifact, though the stimulus

train did continue through acquisition of the Rhod-2 images. Thus, Rhod-2 images

provided details of both mitochondrial calcium following the tetanus and the

approximate myoplasmic calcium. Consequently, relative changes in mitochondrial

Ca2+ in response to stimulation were expressed as a ratio of normalized I-band

fluorescence to normalized A-band fluorescence. For longitudinal mitochondria,

profiles were centered over the A-band and reported as a ratio to A-band signal.
 78

Statistical significance versus resting fluorescence was determined with Kruskal-

Wallis one way ANOVA on ranks with Dunn’s post test. For comparison of

fluorescence in the presence and absence of EGTA, a Mann-Whitney rank sum test

was utilized. Statistical significance was accepted at p < 0.05.

2.3. RESULTS

2.3.1. Mitochondria Accumulate Calcium Following Tetanic Stimulation

We measured mitochondrial calcium uptake in single isolated FDB skeletal

muscle fibers from young (1 month of age) and adult (4 months of age) mice using

the high-affinity (in vitro Kd ~570 nM) calcium indicator Rhod-2. Rhod-2 has

traditionally been the fluorescent indicator of choice for measuring mitochondrial

calcium uptake because its cationic nature directs dye accumulation to mitochondria.

However, several published studies of striated muscle cells report a significant level

of cytosolic Rhod-2 fluorescence, confounding measurements of mitochondrial

calcium. In these studies, selective mitochondrial loading was attained by: 1) cold

loading/warm incubation procedure;241 2) lengthy loading time (90-120 min)

following by an extended wash period to eliminate cytoplasmic dye;36,37,140,141 or 3)

cell permeabilization followed by cytosolic washout.212 Despite my optimization of

the loading procedures, FDB fibers exhibited a significant level of cytosolic Rhod-2

signal. Moreover, sarcolemmal permeabilization collapses the membrane potential,

preventing electrical stimulation of SR calcium release. Accordingly, Rhod-2/AM

was loaded into intact FDB skeletal muscle fibers at room temperature for a limited
 79

amount of time. I took advantage of residual myoplasmic Rhod-2 signal to normalize

mitochondrial fluorescence as explained in the Methods section.

At rest, Rhod-2 fluorescence in both young and adult FDB fibers exhibited

transverse A-band (myoplasmic and out-of-focus mitochondrial) fluorescence

alternating with a brighter I-band signal (Figure 2.1). In the young fibers, Rhod-2

rarely exhibited longitudinal fluorescence, though longitudinal mitochondrial clusters

were clearly visible in the MTG channel. Additionally, the resting I-band Rhod-2

signal in some fibers did not colocalize with MTG fluorescence (data not shown),

suggesting a contribution of non-specific Rhod-2 binding of I-band proteins to the

diffuse I-band signal. Agents to collapse the membrane potential (e.g. FCCP, CCCP)

to inhibit mitochondrial calcium uptake were not employed to gauge the contribution

of non-specific signal to I-band measurements because mitochondrial uncouplers

have been reported to increase resting myoplasmic calcium and alter electrically-

evoked myoplasmic calcium transients.45 Instead, a stimulation protocol was

designed to maximize the appearance a discrete Rhod-2 signal, comparable to the

disposition of the MTG signal.

Low frequency (1 Hz) stimulation of adult fibers triggered a general increase

in Rhod-2 fluorescence, consistent with an increase in myoplasmic calcium with no

significant mitochondrial calcium accumulation (Figure 2.1). By contrast, a single

train of high frequency stimulation (500 ms, 100 Hz) produced a uniform increase in

A-band fluorescence (Figure 2.1 and 2.4B and D) with a heterogenous increase in I-

band fluorescence (Figures 2.1 and 2.4A). Specifically, the Rhod-2 signal in certain
 80

areas of the fibers exhibited transverse double rows of fluorescence, indicating

significant mitochondrial calcium uptake. In other areas, there was a parallel increase

in both I-band and A-band fluorescence, but the I-band signal remained diffuse.

Similarly, Rhod-2 fluorescence in young fibers exhibited the same heterogeneity

following a single high frequency train (Figure 2.4A). Calcium uptake in intermittent

regions of longitudinal and triadic mitochondria was apparent in young fibers, as

well. Thus, subsequent experiments in both young and adult FDBs utilized a high

frequency stimulation protocol to examine mitochondrial calcium uptake following

SR calcium release, and all measurements were made in areas of discrete Rhod-2

signal.

Muscle fibers were stimulated with a series of 40 tetani at 2.5 s intervals

(start-to-start) to evoke prolonged SR calcium release (Figure 2.2A). Measurements

of global calcium with the low affinity indicator Mag-Fluo-4 revealed that a single

high frequency train evoked rapid SR calcium release, with an initial rise time of a

few milliseconds (3.86 ± 0.02 ms) (Figure 2.2B). There was a summation of the first

few pulses during the train such that peak calcium levels (1.26 ± 0.03 ∆F/F0) (Figure

2.2C) were reached within a few tens of milliseconds and remained high for the

duration of the stimulus. Indicative of rapid myoplasmic calcium clearance, Mag-

Fluo-4 fluorescence returned to baseline within a couple hundred milliseconds

following the end of the stimulation train (Figure 2.2B). Subsequent tetani elicited

largely the same response as the first train, with a tendency for decreased peak

amplitude after ~15-20 tetani (Figure 2.2D). Importantly, there was no significant
 81

increase or decrease in baseline fluorescence from the first to the last tetanus (Figure

2.2E).

As mentioned above, triadic mitochondria in young and adult FDB fibers

accumulated calcium following the first tetanus (Figure 2.4A and C). The increase in

I-band/A-band fluorescence after the first tetanus was similar between both young

and adult fibers (1.17 ± 0.04 and 1.19 ± 0.04, respectively). There was a trend for

continued increase in mitochondrial calcium uptake with the next five to ten tetani,

although the increases were not significantly different from the initial rise after the

first tetanus. Regardless, the signal remained elevated throughout tetanic stimulation

and at 1 minute following the last stimulus train (Figure 2.4A and C). On the other

hand, the Rhod-2 I-band/A-band ratio had returned near baseline levels by 20 minutes

(1.04 ± 0.01 and 1.06 ± 0.02 for 1 and 4 month fibers, respectively). Moreover, Fluo-

4 measured in sequence with Rhod-2 in a subset of these experiments indicated that

myoplasmic calcium was back to resting levels at 1 minute following the last tetanus

(Figure 2.3A and B). In addition, we know from the Mag-Fluo-4 studies that

myoplasmic calcium rapidly returns to baseline following each tetanus (Figure 2.2B).

Taken together, this data supports the conclusion that the increase in I-band

fluorescence in areas of discrete Rhod-2 signal reflects prolonged mitochondrial

calcium uptake and not myoplasmic calcium elevations.

Like triadic mitochondria in young fibers, a significant change in longitudinal

fluorescence relative to A-band fluorescence was also observed after a single tetanus

(1.22 ±0.09) (Figure 2.4A and E). The change in longitudinal fluorescence followed
 82

a similar time course as I-band fluorescence, remaining high for the length of the

stimulation protocol and decreasing gradually to baseline following the final tetanus

(Figure 2.4E). The magnitude of change with tetanic stimulation was similar between

triadic and longitudinal mitochondria. However, direct comparisons were not made

between the two as clear longitudinal mitochondria were rarely in the same focal

place as triadic mitochondria in the same images, artificially skewing measurements

of longitudinal fluorescence toward higher values than triadic measurements.

Interestingly, there was no detectable change in TMRE fluorescence in either

young or adult FDB fibers during the tetanic stimulation protocol (Figure 2.3A and

C). In other words, mitochondrial calcium uptake in my experiments did not induce a

significant sustained depolarization of mitochondria. Additionally, retention of the

TMRE dye and response to a single twitch at the end of the tetanic stimulation

protocol indicated that both the mitochondrial and sarcolemmal potentials were intact

at the conclusion of the experiment. Thus, the observed mitochondrial response to

SR calcium release cannot be attributed to calcium overload and subsequent cell

death. It is worth noting that any rapid or transient changes in mitochondrial

membrane potential would have escaped detection with the relatively slow and

intermittent image acquisition utilized in this study.

2.3.2. EGTA Reduces Mitochondrial Calcium Uptake During Tetanic Stimulation

To test the privileged nature of mitochondrial calcium uptake during tetanic

stimulation, Rhod-2 fluorescence was measured in fibers loaded with the calcium
 83

chelator EGTA-AM. EGTA has relatively slow kinetics, and thus, is unable to buffer

calcium within the microdomain (i.e. nanometers from the calcium release source). If

the mitochondrial calcium uptake mechanism is located closer than a couple hundred

nanometers of the microdomain,180 EGTA will have a limited effect. In my

experiments, one criterion for choosing fibers to image was twitch with a single, low

frequency stimulus. Therefore, I loaded a relatively low concentration of EGTA-AM

to only partially reduce myoplasmic calcium transients. Following loading with 25

µM EGTA-AM, the peak myoplasmic calcium level after a single tetanus was

significantly reduced (1.26 ± 0.03 ∆F/F0 and 0.75 ± 0.02 ∆F/F0 in the absence and

presence of EGTA, respectively) (Figure 2.2C). The initial rise phase and the decay

exhibited similar kinetics as in the absence of EGTA (Figure 2.2B).

Similar to resting fibers in the absence of EGTA, both young and adult fibers

exhibited transverse rows of diffuse I-band fluorescence. Within the first tetanus,

discrete transverse rows of mitochondrial fluorescence were visible. Just as before,

mitochondrial calcium uptake peaked after the first few tetani, remained elevated for

the duration of tetanic stimulation, and gradually decreased to resting levels after the

last tetanus (Figure 2.4C). In young fibers, mitochondrial calcium uptake in neither

triadic mitochondria (Figure 2.4C) nor longitudinal mitochondria (Figure 2.4E) was

significantly affected by EGTA. On the other hand, mitochondrial calcium uptake in

triadic mitochondria of adult fibers after the first tetanus was significantly (p < 0.01)

lower in the presence of EGTA (1.19 ± 0.04 and 0.96 ± 0.03, respectively). This

decrease was moderately significant (0.05 > p > 0.01) following subsequent trains.
 84

2.4. DISCUSSION

My initial studies revealed that with a single low frequency twitch,

mitochondria in FDB fibers did not take up detectable calcium. Therefore, I utilized

high frequency tetanic stimulation to maximize SR calcium release, anticipating

significant mitochondrial calcium uptake. Given that the typical nerve impulse

frequency for fast-twitch muscle is ~50-100 Hz,104 high frequency stimulation is a

more physiological condition under which to observe mitochondrial calcium uptake.

In fact, Lännergren and colleagues employed a similar paradigm to examine

mitochondrial calcium uptake in intact murine FDB fibers.140 In their study, single

FDB fibers were stimulated at 70-80 Hz for 350 ms at 4 s intervals, then at 3 s

intervals, and finally at 2.5 s intervals until tetanic force declined to 40% of rest (up to

60 tetani). Mitochondrial calcium was measured with Rhod-2 fluorescence before,

during and after the fatiguing stimulation. Yet, the authors reported that

mitochondrial calcium uptake was negligible during tetanic stimulation.140 Based

upon this study, I began stimulating fibers with 350 ms trains at 70 Hz at 2.5 s

intervals. In contrast to Lännergren et al.,140 I observed an increase in mitochondrial

calcium within the first few trains. Ultimately, after some optimization of the

protocol to maximize SR calcium release and subsequent mitochondrial calcium

uptake, I set stimulation at 500 ms, 100 Hz trains given every 2.5 s for a total of 40

tetani. Certainly, the differences in stimulation paradigms between my experiments

and the published study are minor. Consequently, this experimental variation cannot

entirely account for the conflicting results.

 85

Alternatively, differences in the Rhod-2 fluorescence in quiescent fibers may

explain the apparent discrepancies. I report diffuse Rhod-2 I-band fluorescence at

rest. On the other hand, Lännergren et al. described defined transverse rows of Rhod-

2 fluorescence in unstimulated fibers with very little myoplasmic signal.140 This

signal is not unlike the discrete transverse mitochondrial fluorescence I see after

tetanic stimulation. Conceivably, variations between the mouse strains used or the

fiber isolation procedure, namely manual dissection140 versus enzymatic dissociation

(see Section 1.2.1) could result in the differing Rhod-2 signal at rest. Regardless, the

data suggests that mitochondrial resting free calcium was elevated in the Lännergren

study. Consequently, any mitochondrial calcium uptake they observed would be

small relative to the change in mitochondrial calcium in our studies.

The significant increase in mitochondrial free calcium I observed following a

tetanic train, but not a single twitch, implies that sustained SR calcium release is

necessary for significant mitochondrial calcium uptake. In the future, it will be

essential to examine a spectrum of stimulation frequencies, with trains of varying

length and timing. Essentially, this would enable selective titration of the duration

and magnitude of SR calcium release. This type of systematic study will provide

detailed information about the conditions of SR calcium release that stimulate

mitochondrial calcium uptake. Furthermore, a more detailed analysis may reveal a

significant difference in magnitude or time course of mitochondrial calcium uptake in

young versus adult triadic mitochondria. In the present study, no such differences

were detected. It is possible that, regardless of developmental stage, I-band localized

 86

mitochondria are equivalent in terms of calcium uptake. In addition, dosing SR

calcium release would provide evidence for (or against) a threshold calcium for

mitochondrial calcium uptake. If a threshold exists, it may differ between young and

adult FDB skeletal muscle fibers. Specifically, a progressive decrease in uptake

threshold could accompany maturation of mitochondrion-CRU pairing throughout

postnatal development.

Showing an inverse relationship between a mitochondrial calcium uptake

threshold and mitochondrion-CRU pairing would support that mitochondria respond

to microdomain calcium. On the other hand, failure to see a correlation between

mitochondrial calcium uptake and mitochondrial localization with respect to triads,

would, instead, indicate that mitochondria are responding to global calcium. The

limited effect of EGTA on calcium uptake in triadic mitochondria of young fibers

supports the former. However, the trend in adult fibers for reduced mitochondrial

calcium uptake in the presence of EGTA intimates that mitochondria do respond to

global calcium. Perhaps, simply dampening myoplasmic calcium with a low EGTA

concentration is not sufficient to eliminate mitochondrial calcium uptake. Thus, these

results cannot adequately support or refute privileged SR-mitochondrial calcium

signaling during EC coupling. In fact, the true test of highly privileged mitochondrial

calcium uptake following SR release is in the inability of the fast calcium buffer

BAPTA to reduce mitochondrial calcium uptake.179,180 The fast kinetics of BAPTA

allow it to confine the calcium microdomain, chelating calcium within a few tens of

nanometers from the source of release.179,180 Thus, the effect of BAPTA on

 87

mitochondrial calcium uptake could reveal important information about the proximity

of mitochondrial calcium uptake sites with respect to CRUs and whether

mitochondria are indeed responding to microdomains or global calcium.

Moreover, opposing effects of calcium chelators on triadic and longitudinal

mitochondria within the same muscle fiber would provide more definitive proof of

the intimacy of SR-mitochondrial association. If mitochondrion-CRU pairing is

essential for significant mitochondrial calcium uptake during EC coupling, then

EGTA and BAPTA would be equally effective in reducing longitudinal mitochondrial

calcium uptake, whereas only BAPTA would effectively reduce triadic mitochondrial

calcium uptake. In young FDB fibers with significant populations of both

longitudinal and triadic mitochondria, I was unable to resolve differences between the

two. In particular, differences in the focal planes of longitudinal versus triadic

mitochondria prevented direct comparisons within the same fiber in this limited set

data set. Not to mention, the longitudinal clusters were often concentrated beneath the

sarcolemma. Thus, mitochondrial calcium accumulation from calcium influx during

prolonged activation cannot be ruled out.52,153 Future experiments conducted in

calcium-free solution would reveal the contribution of calcium influx to

subsarcolemmal mitochondrial calcium uptake.

In conclusion, I have shown that both triadic and longitudinal mitochondria in

young and adult fast-twitch skeletal muscle fibers accumulate calcium with tetanic

stimulation. These results justify a more thorough investigation to determine the

exact conditions under which mitochondria take up calcium. Equally important,

 88

subsequent studies should examine mitochondrial calcium uptake in fast-twitch

oxidative (e.g. extensor digitorum longus) and slow-twitch muscle fibers, which are

known to have significant populations of longitudinal and triadic mitochondria even

in adult muscle. These studies, when paired with experimental use of calcium

chelators and high resolution ultrastructure studies, will provide more information

regarding orthograde SR-mitochondrial calcium signaling. Furthermore, each of

these fiber types differs in their reliance on glycolysis versus oxidative

phosphorylation. Thus, any variation in mitochondrial calcium uptake among the

different fibers types will give insight into the importance of mitochondrial calcium

uptake for ATP production, namely excitation-metabolism coupling.

 89

Figure 2.1. Rhod-2 Fluorescence in FDB Skeletal Muscle Fibers

A) Representative confocal images of MitoTracker® Green FM (MTG) and Rhod-2

fluorescence in FDB skeletal muscle fibers obtained 4 month-old mice before and

after electrical stimulation. B) MTG (left) and Rhod-2 (right) fluorescence along the

line of interest marked in A.

 90

Figure 2.2. Mag-Fluo-4 Measurements of Myoplasmic Calcium

A) Tetanic stimulation protocol: 5 ms, 8 V square-wave pulses at 100 Hz for 500 ms

(left) at 2.5 s intervals for a total of 40 tetani (right). Filled squares indicate the time of

image acquisition. B) Representative electrically-evoked myoplasmic calcium transients

in FDB fibers obtained from 1 month-old mice in the absence (upper) and presence

(lower) of EGTA. C) Mean (± SE) peak fluorescence (∆F/F) of tetanic calcium

transients in the absence and presence of EGTA. D and E) Mean (± SE) peak

fluorescence (∆F/F0) (D) and resting fluorescence (F0) (E) of each tetanic train.
 91

Figure 2.3 Mitochondrial Membrane Potential is Stable During Tetanic

Stimulation

A) Representative confocal images of Fluo-4 (green) and TMRE (red) fluorescence in

FDB fibers obtained from 1 (left) and 4 (right) month-old mice before tetanic

stimulation; after 1 and 40 tetani; and 1 and 20 minutes following the last (40th)

tetanus. Bars: 5 µm. B and C) Mean (± SE) normalized myoplasmic fluorescence

(∆F/F0) (B) and triadic TMRE fluorescence (Normalized FI-band/Normalized FA-band)

during tetanic stimulation at 1 (circles) and 4 (triangles) months.

92
 93

Figure 2.4. Mitochondrial Calcium Increases During Tetanic Stimulation

A) Representative confocal images of Rhod-2 fluorescence in FDB fibers obtained

from 1 (upper) and 4 (lower) month-old mice before tetanic stimulation; after 1, 5,

10, and 40 tetani; and 1 and 20 minutes following the last (40th) tetanus. Bars: 5 µm.

Normalized A-band fluorescence (Ft/Ft=0) (B) and Mean (± SE) normalized triadic

Rhod-2 fluorescence (Normalized FI-band/Normalized FA-band) (C) during tetanic

stimulation at 1 (circles) and 4 (triangles) months in the absence (filled symbols) and

presence (open symbols) of EGTA. Normalized A-band fluorescence (Ft/Ft=0) (D)

and mean (± SE) normalized longitudinal Rhod-2 fluorescence (Normalized

Flongit/Normalized FA-band) (E) during tetanic stimulation at 1 month in the absence

(filled circles) and presence (open circles) of EGTA.

 94

CHAPTER 3: MITOCHONDRIAL SUPPRESSION OF LOCAL CALCIUM

RELEASE
 95

3.1. RATIONALE

Spontaneous SR calcium release events, termed calcium sparks, are extremely

rare in adult mammalian skeletal muscle under basal conditions. Potential

mechanisms to explain the absence or suppression of calcium sparks include direct

mitochondrial calcium buffering and redox modulation of the microenvironment

surrounding SR calcium release sites by energized and functionally intact

mitochondria.117,118 I hypothesized that exclusive positioning of mitochondria to the

CRUs is a prerequisite for calcium spark suppression by mitochondria. Moreover, as

results presented in Chapter 1 found that mitochondrial positioning adjacent to sites

of calcium release differed dramatically during postnatal development, calcium spark

suppression was expected to be reduced early during postnatal development.

Accordingly, the present study compared the incidence and properties of osmotic

shock-induced calcium sparks in skeletal muscle fibers throughout postnatal

development. I expected that increased mitochondria positioning adjacent to CRUs

during postnatal development would result in greater suppression of calcium sparks.

3.2. METHODS

3.2.1. Preparation of Muscle Fibers

Single acutely dissociated FDB muscle fibers were isolated from young (0.5

and 1 month old) and adult (2 and 4 month old) C57/Bl6 mice as described in Section

1.2.1. Fibers were plated in a modified Ringer’s solution (in mM: 140 NaCl, 4 KCl, 1
 96

MgSO4, 1.8 CaCl2, 5 NaHCO3, 10 Glucose, 10 HEPES; pH 7.4, ~300 mOsm) on

glass cover slips and allowed to settle for 20 minutes before dye loading.

3.2.2. Confocal Imaging and Analysis of Calcium Sparks

Fibers were loaded with 10 µM Fluo-4 AM for 60 minutes at room

temperature followed by 10 minutes in dye-free solution. To induce calcium sparks,

fibers were immersed in hypertonic Ringer (in mM: 140 NaCl, 4 KCl, 1 MgSO4, 50

CaCl2, 5 NaHCO3, 10 Glucose, 10 HEPES; pH 7.4, ~450 mOsm). With the focal

plane centered along fiber periphery, a series of line scans (1024 lines, 47.7 µm/line,

2.12 s/line) parallel to the long axis of the fiber (i.e. spanning multiple sarcomeres)

were acquired. To enable comparison between different fibers, images were recorded

using identical laser power, photomultiplier sensitivity, and were processed using

identical values for contrast and brightness. Images were analyzed offline using

CaSparks automated detection software kindly provided by Drs. W. Melzer and D.

Ursu (Department of Applied Physiology University of Ulm, Ulm, Germany).

Detection limits were set broadly to identify events with amplitudes (A) ranging from

0.2 to 6 ∆F/F, full width at half-maximal amplitude (FWHM) from 0.5 to 50 µm, rise

time (R) from 0.5 to 100 ms, and full duration at half-maximal amplitude (FDHM)

from 0.5 to 1000 ms. Spark mass was calculated as A*1.206*FWHM3, according to

Hollingworth et al.113 Statistical significance (p < 0.01) was determined with

Kruskal-Wallis one-way ANOVA on ranks with Dunn’s post test.

 97

3.3. RESULTS

3.3.1. Spatiotemporal Properties of Osmotic Shock-Induced Calcium Sparks Vary

in an Age-Dependent Manner.

I hypothesized that mitochondrial localization adjacent to CRUs would

facilitate inhibition of local calcium release at the triad. To test this hypothesis in

single intact FDB fibers, the incidence and properties of calcium sparks, revealed by

exposure to hypertonic Ringer, were compared at different times during postnatal

skeletal muscle development (0.5, 1, 2, and 4 months) that were shown in Chapter 1

to exhibit marked differences in mitochondrial-CRU colocalization. Subsequent to

exposure to hypertonic Ringer, FDB fibers of all ages exhibited spontaneous local

calcium release events (Figure 3.1). Activity persisted for the hour of

experimentation following osmotic shock and occurred primarily at the periphery of

the fibers. Because the external solution contained calcium, I cannot exclude the

possibility that some sparks were in fact calcium influx through membrane tears

caused by osmotic shock. However, the quantal nature of the observed events argues

against membrane tears. Furthermore, triads are found within a few microns of the

sarcolemma (Figure 3.5, Table 3.2) and in the few number of line scans where a clear

Fluo-4 banding pattern was observed, events did occur in the expected triadic position

(low fluorescence I-band19).

Spark morphology was variable throughout development. At all ages, brief

(~20-30 ms) local calcium release events (classic Ca2+ sparks) were observed (Figure

3.1A and B) with time courses similar to the calcium sparks identified in other
 98

mammalian skeletal muscle preparations.35,117,118,250,253 In addition, longer events

(>100 ms), analogous to so-called calcium “bursts”250 were also detected (Figure

3.1A and C). To examine a broad range of events, an upper limit was set on both rise

time (≤ 100 ms) and FDHM (≤ 1000 ms).

The events were segregated into short (rise time < 30 ms and/or FDHM < 100

ms) and long (rise time > 30 ms and/or FDHM > 100 ms) events. The percentage of

short events was greatest at 0.5 month (61% of total events) and smallest at 1 month

(41%). The total population of events was split 50/50 between short and long at 2

and 4 months (Figure 3.2A). However, the parameters varied with age within the

population of short events to the same degree as within the population of long events.

Consequently, all subsequent analyses were conducted on the entire population of

detected events, and all events from here on will be generally referred to as “Ca2+

sparks”.

Analysis of average (± SE) Ca2+ spark amplitude, spatial width, and mass are

presented in Figure 3.3. Mean peak Ca2+ spark amplitude was significantly reduced

in 0.5 month fibers (0.34 ± 0.01 ∆F/F) compared to events observed in fibers isolated

from 1 (0.41 ± 0.01 ∆F/F), 2 (0.40 ± 0.01 ∆F/F), and 4 (0.41 ± 0.01 ∆F/F) month-old

mice, which exhibited similar peak amplitudes (Figure 3.3A, Table 1). Likewise,

events at 1, 2, and 4 months exhibited similar FDHM (48.26 ± 2.67 ms, 47.26 ± 2.08

ms, and 49.62 ± 1.83 ms, respectively) which were greater than that observed at 0.5

months (33.66 ± 1.94 ms) (Figure 3.2C, Table 1). A similar trend (an increase during

early postnatal development) was not observed for either Ca2+ spark rise time or
 99

FWHM. Rather, rise time and FWHM peaked at 1 month and then were reduced

thereafter (Table 3.1). Specifically, rise time at 1 month (38.22 ± 1.04 ms) was

significantly longer than 0.5, 2, and 4 month events (29.54 ± 0.79 ms, , 33.28 ± 0.71

ms, and 33.87 ± 0.54 ms, respectively) (Figure 3.2B, Table 3.1). In addition, the

FWHM was greater at 1 month (1.48 ± 0.02 µm) than that observed at 0.5, 1 or 2

months (1.35 ± 0.02 µm, 1.36 ± 0.02 µm, and 1.30 ± 0.01 µm, respectively) (Figure

3.3B, Table 3.1). As a result, spark mass (amplitude*1.206*FWHM3 from 113

) was

greatest at 1 month (1.8 ± 0.2 µm3, 2.7 ± 0.2 µm3, 2.1 ± 0.1 µm3, and 1.8 ± 0.1 µm3

for 0.5, 1, 2, and 4 months, respectively) (Figure 3.3C, Table 3.1).

Finally, raw Ca2+ spark frequency followed the identical pattern of both

amplitude and FDHM, being lowest at 0.5 month (76.12 ± 3.72 events/min) (Figure

3.4A). Higher Ca2+ spark frequencies observed in FDB fibers of 1, 2 and 4 month-old

mice (115.02 ± 9.05 events/min, 110.14 ± 5.70 events/min, and 107.81 ± 4.27

events/min, respectively) (Figure 3.4A, Table 3.1 ) most likely reflect an increase in

the CRU number and density that occurs during postnatal development (Tables 1.1

and 3.2). Following normalization to CRU density at each developmental stage

determined from EM analysis (Table 1.1), normalized Ca2+ spark frequency was

found to decrease progressively during postnatal development (Figure 3.4B, Table

3.1). Fibers isolated from 0.5 month-old mice exhibited the greatest frequency of

events per density (3.62 ± 0.18 events/min/CRU/100 µm2) followed by 1, 2, and 4

months (2.67 ± 0.21 events/min/CRU/100 µm2, 1.60 ± 0.08 events/min/CRU/100

µm2, and 1.23 ± 0.05 events/min/CRU/100 µm2, respectively). Importantly, there

 100

was a clear linear correlation between the tether frequency29 and Ca2+ spark frequency

per CRU density (Figure 3.4C).

3.3.2. Osmotic Shock Induces Subsarcolemmal Changes

Preservation of MitoTracker® Red CMXRos (MTR) fluorescence pattern in

FDB fibers isolated from 0.5 month-old mice upon exposure to hypertonic Ringer

(Figure 3.5C) indicate that osmotic shock treatment used to trigger Ca2+ sparks does

not grossly alter mitochondrial localization. In addition, electron microscopy analysis

revealed no gross ultrastructural changes in the interior of the fiber following osmotic

shock fibers of either 0.5 or 4 month FDB fibers in the interior of the fiber (data not

shown). On the other hand, osmotic shock did induce minor changes in the

subsarcolemmal region (within 1 µm of the surface membrane) (Figure 3.5A and B).

In particular, exposure to hypertonic Ringer induced swelling of transverse tubules

(T-tubules) and caveolae just beneath the surface membrane, the extent of which was

variable within a given field of view. Importantly, osmotic shock treatment produced

similar changes in FDB fibers from both 0.5 and 4 month old mice. The only major

difference between the two ages was a higher percentage of fibers with

subsarcolemmal and intermyofibrillar clusters of mitochondria (Table 3.2), consistent

with the increase in intermyofibrillar mitochondrion-CRUs pairs described in Chapter

1.29
 101

3.4. DISCUSSION

Since the original identification of calcium sparks in cardiac myocytes,51 these

spontaneous, spatially restricted calcium release events have been detected in many

different cell types including intact smooth muscle181 and amphibian skeletal

muscle.130,242 Uniquely, calcium sparks in arterial smooth muscle provide localized

calcium release to selectively activate large-conductance Ca2+-activated K+ channels

(BK channels). Subsequent K+ efflux through BK channels hyperpolarizes cells,

triggering vasodilation.181 In effect, subsarcolemma Ca2+ sparks in arterial smooth

muscle permit SR calcium release to assume a highly localized regulatory function

rather than triggering muscle contraction. By contrast, calcium sparks of mammalian

cardiac and amphibian skeletal muscle represent elementary units of the global

calcium transients evoked during EC coupling.51,131 Extrapolation of macroscopic

calcium from single calcium spark properties is made possible by the homogeneity of

sparks and the relatively high frequency of spontaneous events in cardiac and

amphibian skeletal muscle.66

On the other hand, spontaneous calcium sparks in intact adult mammalian

skeletal muscle are extremely rare,60 though they can be unmasked by sarcolemmal

permeabilization, disruption of mitochondrial function, osmotic shock, and strenuous

exercise. Currently, there are three proposed mechanisms to explain the absence or

suppression of calcium sparks in adult mammalian skeletal muscle under basal

conditions. First, adult mammalian skeletal muscle expresses a single ryanodine

receptor isoform, RyR1. On the other hand, amphibian skeletal muscle expresses
 102

both RyR1 (RyRα) and RyR3 (RyRβ) isoforms, suggesting that RyR3 accounts for

the presence of calcium sparks in amphibian skeletal muscle.131 Likewise, Ca2+

sparks are readily detectable in mammalian diaphragm, which expresses both RyR1

and RyR3, and following overexpression of RyR3 in mammalian FDB muscle

fibers.194 Moreover, spark frequency decreases concurrently with decreased RyR3

expression during the transition of myotubes into myofibers, though Ca2+ sparks are

also observed in RyR3-null myotubes,254 indicating that RyR3 supports, but is not

required for Ca2+ spark activity.131

Second, tight coupling of DHPR and RyR1 at triad junctions in mammalian

skeletal muscle may ensure exclusive control of SR calcium release by the voltage-

sensor (i.e. DHPR), thereby preventing spontaneous calcium release events in resting

skeletal muscle cells that lack RyR3. In amphibian muscle, voltage-induced calcium

release (VICR) initiated by the DHPR can activate lone RyRs that are not coupled to

a voltage sensor as a result of calcium-induced calcium release (CICR). CICR can

also occur independent of VICR and is responsible for spontaneous sparks in

amphibian skeletal muscle. On the other hand, CICR is not a significant means of

release control in mammalian skeletal muscle, presumably due to tight DHPR-RyR1

coupling, and thus, spontaneous calcium release is rare. Additional evidence that the

DHPR-RyR1 interaction suppresses mammalian sparks is the detection of sparks in

skeletal muscle myotubes101,211 and dedifferentiated skeletal muscle.35 In these cell

types, the T-tubular network lacks the structure of adult muscle. Further, osmotic

shock, a technique employed to reveal sparks in intact adult mammalian skeletal

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muscle may physically disrupt triads, and thus, the tight DHPR-RyR1 inhibitory

interaction. In contrast, mechanical skinning, another technique that uncovers

calcium sparks in adult mammalian skeletal muscle, does not compromise DHPR-

RyR1 coupling. Thus, while the DHPR-RyR1 interaction likely contributes to spark

suppression in adult mammalian skeletal muscle, but it is not sufficient. Indeed,

experiments conducted in control and DHPR-null (dysgenic) myotubes revealed that

the DHPR plays a critical role in reducing the rate of spontaneous Ca2+ spark

generation.268

Clearly, additional mechanisms are needed to explain the absence of calcium

sparks in mammalian skeletal muscle. Most recently, Isaeva et al.117,118 proposed that

mitochondria contribute to spark suppression. In particular, they argued that

perforation of the sarcolemma damages mitochondria and thus, relieves inhibition of

calcium sparks. Their initial studies clearly demonstrated that Ca2+ spark frequency

is highly dependent on mitochondrial respiration.117 Specifically, stimulating

respiration by providing substrates for the tricarboxylic acid (TCA) cycle reversibly

reduced spark frequency in saponin-permeabilized fibers. Conversely, inhibition of

respiration with antimycin A or dissipation of mitochondrial membrane potential with

FCCP and oligomycin increased calcium spark frequency. In addition, blocking

mitochondrial calcium uptake with Ru360 increased the incidence of sparks,

suggesting that mitochondrial inhibition of sparks involves calcium buffering.

Though not yet identified in skeletal muscle, miniature transient elevations in

mitochondrial matrix calcium (Ca2+ marks) elicited by calcium sparks have been
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identified in cultured cardiac cells.188 Because mitochondria are so closely positioned

(~130 nm) to sites of calcium release in adult mammalian skeletal muscle,29 it is

possible that mitochondrial calcium uptake directly inhibits junctional CICR and thus

Ca2+ spark activity

However, until Ca2+ marks are described in skeletal muscle, alternative

mechanisms for spark inhibition must be considered. For example, mitochondria may

indirectly regulate SR calcium release via their capacity to generate ATP and ROS.

Indeed, Isaeva and colleagues118 showed a correlation between mitochondrial content,

redox potential, and Ca2+ spark development in permeabilized skeletal muscle fibers.

In their study, the onset of spark activity following permeabilization was slowest in

mitochondria-rich oxidative muscle fibers and significantly faster in mitochondria-

poor glycolytic fibers. Mitochondrial substrates and ROS scavengers also delayed the

appearance of sparks, whereas oxidative insult (e.g. H2O2) accelerated spark

development following sarcolemmal permeabilization. Taken together, the findings

of Isaeva et al. provide evidence that preservation of mitochondrial function is a

factor in calcium spark inhibition in mammalian skeletal muscle, particularly in

permeabilized muscle fibers.

Given the above discussion, I hypothesized that close mitochondrial-SR

juxtaposition is a critical determinant of mitochondrial inhibition of Ca2+ spark

suppression in adult mammalian skeletal muscle. Specifically, tight SR-

mitochondrial association would be expected to create a spatially confined signaling

domain that facilitates both direct calcium buffering of sparks by mitochondria and
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ATP/ROS mediated effects on the release process. Moreover, longitudinally

clustered mitochondria, which are abundant early in postnatal development, would be

predicted to exhibit a more limited capacity to suppress calcium sparks due to their

more remote distance from sites of release. Indeed, I find a progressive decrease in

spark frequency per CRU density from 0.5 to 4 months, a time during which we know

that both the number of mitochondria and percent of mitochondria localized to the I-

band increases (see Chapter 1). The decrease in the propensity for a given CRU to

spark concurrent with increase mitochondrion-CRU pairing is consistent with

mitochondrial spark inhibition. Notwithstanding, spark incidence and morphology

likely reflects a balance of both mitochondrial localization and CRU development.

For instance, the increase in raw frequency of events from 0.5 to 1 month reflects a

burst in CRU density during this time. Further, higher mean peak spark amplitude

and longer FDHM at 1, 2, and 4 months likely results from CRU maturation. That

there is no additional increase in either parameter from 1 to 4 months is not surprising

since the greatest overall triadic structural changes occur between 0.5 and 1 month.

Thus, at a certain point of development (~1 month), CRUs are, in effect, equivalent in

terms of spontaneous release event properties induced by osmotic shock.

Interestingly, trends in the other spatiotemporal properties indicate that the 1

month postnatal stage represents a critical transitional time point. Specifically, rise

time is slowest and both FWHM and spark mass are maximal at 1 month.

Essentially, CRU development in 1 month fibers has outpaced mitochondrion-CRU

targeting, resulting in high amplitude and long events with a large spatial spread.
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Yet, spark frequency per CRU continues to decrease in parallel with increased

mitochondrion-CRU pairing. Finally, at 2 and 4 months, mitochondrion-CRU pairing

is balanced with the level of CRU maturation. Consequently, sparks exhibit no

further changes in amplitude and duration. Because mitochondrion-CRU pairing

increases further from 2 to 4 months, Ca2+ sparks are more spatially restricted and

less frequent than that observed in younger fibers. It will be important for future

experiments to address the following questions by more precisely correlating

mitochondrial and Ca2+ spark location in the same cell. For example, do areas of

repeated spark activity correspond to areas that lack mitochondrial targeting? Is there

a link between Ca2+ spark spatiotemporal properties and mitochondrial orientation?

That is, do briefer sparks occur in regions containing triadic mitochondria, whereas

longer duration calcium “bursts” predominante in areas of longitudinal mitochondria?

Clearly, my results pose many intriguing questions with regard to the relationship

between mitochondrion-CRU targeting and calcium spark suppression.

Of course, future work designed to elucidate the mechanism of spark

suppression requires addressing additional changes during skeletal muscle

development that accompany the increase in mitochondrion-CRU targeting. These

factors include changes in DHPR-RyR1 coupling, the complement of proteins in the

junctional release complex (e.g. triadin, junction, calsequestrin), SR calcium store

content, and mitochondrial function. My work indicates that mitochondrial

membrane potential polarization changes from 0.5 to 4 months (Figure 1.3).

Therefore, mitochondrial calcium uptake and respiration are likely to vary during
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postnatal development. In addition, although osmotic shock does not alter

mitochondrion-CRU colocalization, my work does not address potential disruption in

mitochondrial function triggered by osmotic shock that might alter calcium spark

suppression. Conceivably, osmotic shock induces calcium spark activity by

increasing the oxidative environment around CRUs through enhanced ROS

production. Although I do not have direct evidence of enhanced ROS production

with osmotic shock, future work utilizing ROS indicators could be used to test for

increased mitochondrial-derived ROS. These results could directly implicate altered

mitochondrial function in calcium spark genesis in intact mammalian skeletal muscle.

Osmotic shock-induced calcium sparks may also involve factors other than

functional changes in mitochondria. Specifically, the structural changes in caveolae

observed following osmotic shock hint at other possible mechanisms of calcium spark

unmasking. Caveolae are small (50-100 nm) omega-shaped invaginations of the

sarcolemma.96 In striated muscle, the principle component of caveolae is the integral

membrane protein caveolin-3, which serves a variety of functions such as vesicle

trafficking and scaffolding for signal transduction.96 Caveolin-3 interacts with

proteins of the dystrophin-glycoprotein complex, and mutations in caveolin-3 result

in certain forms of muscular dystrophy.96 Additionally, caveolin-3 has been shown to

interact with the glycolytic enzyme phosphofructokinase-M,205 suggesting that

caveolin-3 plays a role in the regulation of energy metabolism. Interestingly, a direct

interaction between caveolin-3 and nNOS (neuronal nitric oxide synthase) results in a

negative regulation of nNOS activity. Conceivably, structural changes in caveolae

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induced by osmotic shock could disrupt this inhibition, leading to an increase in

reactive nitrogen species (RNS) generation. As a consequence, increased RyR1 S-

nitrosylation could result, which has recently been shown to sensitize RyR1 to

activation.81 In short, osmotic shock may trigger an increase in local

oxidative/nitrosative stress that overwhelms mitochondrial scavenging/detoxification

capacity, resulting in direct modification of RyR1 and a subsequent increase in Ca2+

spark activity. Although mitochondria localized adjacent to CRUs would be better

positioned to counter this insult, they may not be sufficient to suppress sparks

altogether, particularly at the periphery where caveoli are located. More importantly,

if the aforementioned hypothesis proves to be correct, study of calcium spark

inhibition could provide insight into more physiological local ROS/RNS signaling

between mitochondria and the SR and the role of this process in some forms of muscle

disease.
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Table 3.1. Calcium Spark Spatiotemporal Properties

All acquired X-t line scan images were processed with CaSparks automated detection

software as described in Methods. Raw frequency was calculated as events/scan and

extrapolated to events/minute. The raw frequency was normalized to the CRU

densities reported in Table 1.1. * p < 0.01 for comparisons with 4 months.
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Table 3.2. Electron Microscopy Analysis of Subsarcolemmal Mitochondria

Measurements for a single set electron micrograph of FDB fibers isolated in isotonic

solution from 0.5 and 4 month-old mice. Mitochondria and CRUs quantified as in

Section 1.2.3.
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Figure 3.1. Osmotic Shock-Induced Ca2+ Sparks and Bursts in FDB Fibers During

Postnatal Development

A) Representative X-t line scan images of Ca2+ sparks in 0.5, 1, 2, and 4 month FDB

fibers. Images were processed using a Gaussian smoothing function and median

filter, followed by background normalization. B and C) Time profiles of each arrow-

marked event in A. B) Short events with time courses less than 50 ms. C) Long

events with time courses greater than 50 ms. Amplitude, rise time, and FDHM (A, R,

and D, respectively) are reported for each event.

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Figure 3.2. Temporal Properties of Osmotic Shock-Induced Calcium Sparks Vary

in an Age-Dependent Manner

A) Percent of short (rise time < 30 ms and/or FDHM < 100 ms, black) and long (rise

time > 30 ms and/or FDHM > 100 ms, white) events. B) Mean (± SE) rise time of

total population of events (short and long). C) Mean (± SE) FDHM of total

population of events (short and long). Statistical significance determined with Fisher

Exact text (A) and Kruskal-Wallis one way ANOVA on ranks with Dunn’s post test

(B and C). * p < 0.01 compared with 4 months.

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Figure 3.3. Ca2+ Spark Mass Peaks at 1 Month During Postnatal Development

A) Mean (± SE) peak Ca2+ spark amplitude. B) Mean (± SE) FWHM. C) Mean (±

SE) Ca2+ spark mass calculated as: amplitude*1.206*FWHM3. * p < 0.01 compared

with 4 months.
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Figure 3.4. Osmotic Shock-Induced Ca2+ Spark Frequency per CRU Density

Decreases with Age

Mean (± SE) raw frequency of Ca2+ sparks (A) and Ca2+ spark frequency normalized

to CRU density (see Table 1.1) (B). * p < 0.01 compared with 4 months. C)

Correlation between normalized Ca2+ spark frequency and tether density (0.8

tethers/100 µm2, 4.0 tethers/100 µm2, 10.6 tethers/100 µm2, and 15.6 tethers/100 µm2

at 0.5, 1, 2, and 4 months, respectively29).

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Figure 3.5. Osmotic Shock Induces Swelling of T-Tubules and Caveolae

A and B) Electron micrographs (longitudinal sections) of 0.5 (A) and 4 (B) month FDB

fibers immersed in isotonic (upper) or hypertonic (lower) solution before fixation. Left

panels depict subsarcolemmal regions. Right panels depict T-tubule profiles within

triads at higher magnification. EM labeling key: caveolae, black arrows; T-tubules,

white arrows. C) Confocal image of MitoTracker® Red CMXRos fluorescence in a

single 4 month FDB fiber before (left) and after (right) exposure to hypertonic solution.
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CHAPTER 4: MITOCHONDRIAL FUNCTION IN SKELETAL MUSCLE

DISEASE
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4.1. RATIONALE

In theory, bidirectional local SR-mitochondrial communication in skeletal

muscle couples activity-dependent calcium signaling with mitochondrial

bioenergetics and redox control. Defects in this local control mechanism could lead

to excessive mitochondrial ROS production that disrupts the cellular redox state in a

manner that contributes to altered Ca2+ release/reuptake during certain forms of

muscle disease. Specifically, I hypothesized that the mitochondrial pathology

associated with Central Core Disease (CCD) reflects defective SR calcium release

that alters SR-mitochondrial calcium signaling and, thus, leads to mitochondrial

dysfunction. Accordingly, I examined mitochondrial localization and membrane

potential in skeletal muscle fibers from knock-in mice heterozygous for a mutation

(Y522S) in RyR1 that, in humans, gives rise to Malignant Hyperthermia (MH) and

CCD coincidence. The expectation was that mitochondrial integrity (i.e. localization,

membrane potential) would be compromised in skeletal muscle fibers from Y522S

mice, as a result of the aberrant calcium release and elevated ROS/RNS production

associated with this mutation.48,81

4.2. METHODS

4.2.1. Preparation of Muscle Fibers

Male-female breeding pairs of heterozygous Y522S knock-in mice (YS),

generated by Dr. Susan Hamilton’s Laboratory at Baylor College of Medicine,48 were

obtained by our laboratory to establish an independent colony of YS mice housed at

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the URSMD (Section 1.2.1). Single acutely dissociated FDB muscle fibers were

isolated from young (0.5 and 1 month-old) and adult (2 and 4 months-old)

heterozygous YS mice as described in Section 1.2.1.

4.2.2. Confocal Imaging and Analysis of Mitochondrial Localization and

Membrane Potential

To assess mitochondrial localization and relative changes in mitochondrial

membrane potential during postnatal development, FDB fibers obtained from 0.5, 1,

2, and 4 month-old mice were loaded with 1.5 µM JC-1 in Ringer’s solution for 20

minutes at 37°C. Fibers were imaged and analyzed offline as described in Chapter 1.

Statistical significance determined with Kruskal-Wallis one-way ANOVA on ranks

with Dunn’s post test or a Mann-Whitney rank sum test for pairwise comparison

where appropriate.

4.2.3. Calculation of Mitochondrial Spacing

To determine sarcomeric spacing, FDB skeletal muscle fibers obtained from

adult (8 month) wild-type (WT) and YS mice were loaded with the potentiometric

mitochondrial indicator, tetramethylrhodamine ethyl ester (TMRE, 10 nM). With the

focal plane positioned at the center of the fiber, TMRE was excited using a 543 nm

(32X attenuation, 605/75 nm emission) to produce an average 47.7 µm x 47.7 µm

image (n = 4). A 5 µm line along the longitudinal axis of the fiber was drawn to

generate a mean XY TMRE fluorescence profile with X values representing fiber

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length and Y values representing the longitudinally averaged fluorescence ratio. The

distance, in microns, between each peak of fluorescence was measured. A 2D blind

deconvolution was applied to each image for display purposes.

4.2.4. Time Lapse Imaging and Analysis of Superoxide Flashes

Time lapse confocal microscopy was used to assess superoxide flashes in WT

and YS FDB fibers expressing a mitochondrial-targeted cpYFP (MT-cpYFP).249

MT-cpYFP was expressed in adult FDB fibers using an electroporation approach

described previously.73 Briefly, wild-type C57/Bl6 mice were anesthetized by

intraperitoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg

acepromazine. FDB muscle was pretreated by intramuscular injection of bovine

hyaluronidase (15 µl, 0.4 U/µl). One hour later, 80 µg of MT-cpYFP in a total

volume of 20 µl 71 mM NaCl was injected using a 30-gauge needle. FDB muscle

was then electroporated using electrodes placed perpendicular to the long axis of the

muscle. Electroporation parameters were 100 V/cm, 20-ms duration, and 20 pulses

delivered at 1 Hz. One-week later, single muscle fibers from electroporated FDB

muscles were isolated by enzymatic dissociation as described above. MT-cpYFP was

excited using a 488 nm (32X attenuation, 515/30 nm emission) laser. Time series

(100 total frames, ~1 frame/s) were acquired using a Nikon Eclipse C1 Plus Confocal

microscope (30 µm pinhole) equipped with a SuperFluor 40X 1.3 NA oil objective

(Nikon Instruments Inc.). Each acquired image was 256 pixels x 256 pixels (0.2

µm/pixel).
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4.3. RESULTS

4.3.1. JC-1 Emission Magnitude is Altered During Postnatal Development in

Y522S Knock-in Mice

Using confocal microscopy, I determined mitochondrial subcellular

localization in single FDB muscle fibers isolated from heterozygous Y522S (YS)

mice at four different stages of postnatal development (0.5, 1, 2, and 4 months).

Early in postnatal development (0.5 month), JC-1 exhibited a predominantly

longitudinal fluorescence, indicative of longitudinal clusters of intermyofibrillar

mitochondria (Figure 4.1A). At 1 month, clear transverse I-band-limited

mitochondria were visible, though most mitochondria remained clustered in the

interior of the fiber. Conversely, the majority of mitochondria in FDB fibers isolated

from 2 month-old mice assumed a transverse localization (Figure 4.1A). In adult

mice (4 months-old), mitochondria were positioned exclusively at the I-band,

consistent with their localization adjacent to CRUs. In short, mitochondria in

heterozygous YS fibers progressively target to the I-band, similar to that observed in

wild-type FDB fibers (see Chapter 1) (Figure 4.1A).

On the other hand, JC-1 ratios of YS fibers differed significantly from WT,

both in the pattern of change throughout development and the mean values (Figure

4.1B). In contrast to WT fibers, the average ratio of JC-1 aggregate (red) to JC-1

monomer (green) fluorescence (Figure 4.1A, right) was the highest in FDBs isolated

from 2 month-old mice (1.37 ± 0.01 at 50% fiber width), followed by 1 (1.20 ± 0.02),

0.5 (1.05 ± 0.02), and 4 months,(0.84 ± 0.01) (Figure 4.1B). Additionally, the mean
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JC-1 ratios of 0.5 and 1 month YS fibers were smaller than the corresponding value

observed in WT fibers (1.21 ± 0.02 and 1.58 ± 0.01 for 0.5 and 1 month,

respectively), whereas the ratios of 2 and 4 month YS fibers were greater than WT

fibers (0.82 ± 0.01 and 0.74 ± 0.01 for 2 and 4 months, respectively).

4.3.2. Mitochondrial Spacing is Similar in WT and YS FDB Fibers

Heterozygous YS fibers exhibited no gross changes in mitochondrial

localization throughout postnatal development that might be suggestive of a

widespread mitochondrial pathology, namely central cores. However, the JC-1

analysis was relatively qualitative. To examine mitochondrial disposition more

quantitatively in adult fibers, I measured the average distance between mitochondria

of adjacent I-bands in single isolated FDB muscle fibers from 8 month-old WT and

YS mice (Figure 4.2A and B). With this approach, a predominance of areas lacking

mitochondrial signal (e.g. cores) would present as a greater mitochondria-

mitochondria distance. Further, measurements of I-band-to-I-band distance would

yield information about sarcomeric spacing. TMRE was chosen as the mitochondrial

probe because it exhibits a very bright and discrete fluorescence pattern.

Both WT and YS fibers exhibit double rows of transverse striations (Figure

4.2A), characteristic of CRU-targeted mitochondria in adult muscle. However,

TMRE fluorescence was quite heterogenous, though the degree of heterogeneity was

the same for both genotypes. For example, TMRE often exhibited punctuate

fluorescence and a number of regions were void of TMRE signal. Several areas of
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each fiber were sampled at random, including these areas of “irregular” fluorescence,

giving rise to the variability of I-band-to-I-band measurements. Importantly, the

middle 50th percentile of values falls within the range of normal sarcomere length (~2

µm). The mean I-band-to-I-band distance was not significantly different between WT

and YS fibers (1.89 ± 0.02 µm and 1.87 ± 0.01 µm, respectively) (Figure 4.2C).

4.3.3. Focal Mitochondrial Damage in YS FDB Fibers

Electron microscopy analysis confirmed the localization of mitochondria at

the I-band, positioned adjacent to CRUs in both WT and YS fibers (Figure 4.3).

However, at this high resolution, clear changes in mitochondrial ultrastructure were

observed in some YS FDB fibers. Specifically, WT mitochondria typically exhibited

a regular rounded or slightly elongated shape, with defined lamellar cristae and an

electron-dense matrix (Figure 4.3A, panels 1-3), though occasionally mitochondria in

WT fibers exhibited membrane myelin figures and disrupted cristae (Figure 4.3A,

panel 4). However, swollen mitochondria with a translucent matrix (Figure 4.3B,

panels 2-4) were frequently observed in YS muscle along with normal mitochondria

(Figure 4.3A, panel 1). In addition, numerous mitochondria appeared to be missing

cristae. Damaged external membranes and vacuole formation were common, as well

(Figure 4.3, panels 2-4). It is possible that these structural abnormalities result in

altered mitochondrial function, such as ROS production. In order to detect focal

changes in mitochondrial ROS production, I utilized a mitochondrial-targeted

superoxide indicator, MT-cpYFP.249 MT-cpYFP is a novel fluorescent probe that

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exhibits an increase in fluorescence emission when oxidized by superoxide. Original

characterization of the probe in cardiomyocytes revealed that it effectively detects

transient increases in superoxide, or superoxide “flashes”, in a single mitochondrion

or a small group of mitochondria.249

Strong colocalization of MitoTracker® Red CMXRos (MTR) and MT-cpYFP

signals confirms mitochondria-specific expression of MT-cpYFP (Figure 4.4A). In

both WT and YS fibers, time lapse imaging revealed transient increases in MT-

cpYFP fluorescence with a fairly large amplitudes (1.4 F/F0 and 1.5 F/F0,

respectively) (Figure 4.4B and C). The flashes were relatively slow, lasting ~ 10 s.

Yet, the flash in the WT fiber appeared to originate from a single mitochondrion,

whereas the flash in the YS fiber clearly covered a network of interconnected

mitochondria. Though preliminary, these data provide proof-of-principle for

detection of superoxide flashes in single isolated FDB fibers and suggest that

mitochondrial function and superoxide production may be altered in skeletal muscle

fibers of YS mice.

4.4. DISCUSSION

The hallmark of CCD is the presence of centrally located, amorphous “cores”

within muscle fibers that are devoid of mitochondrial enzyme activity. Though the

mechanism of core development is unknown, mutations in RyR1 have been identified

as the primary cause of the disease. CCD mutations in RyR1 fall into one of two

categories: 1) C-terminal mutations which result in channels with reduced calcium

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permeation, or excitation-contraction (EC) uncoupled channels; and 2) N-

terminal/central mutations, also associated with MH, which result in leaky channels

with enhanced sensitivity to activation by both voltage and RyR1 agonists.76 Chelu et

al. have generated a knock-in mouse model of one particular N-terminal mutation

(Y522S) that, in humans, results in MH and CCD coincidence.48

The MH phenotype of the Y522S knock-in mouse has been examined

extensively.48,81 Mice heterozygous for the YS mutation in RyR1 survive and breed,

whereas homozygous mice die in utero or at birth. When exposed to triggering

agents, heterozygous mice undergo genuine MH crises, exhibiting whole-body

contractures and elevated core temperature prior to death. In addition, the YS

mutation renders RyR1 more sensitive to activation by ligands (e.g. caffeine) and the

voltage sensor, resulting in an increased calcium leak under basal conditions. At

physiologic temperature, the elevation in myoplasmic calcium stimulates production

of RNS and ROS. Consequently, there is an increase in S-nitrosylation and S-

glutathionylation of the mutant channel. In fact, S-nitrosylation confers heat-

sensitivity to the mutant channel,81 resulting in heat-induced MH-like episodes in YS

heterozygous mice and an increase in heat-induced contractures in vitro.48 At

elevated temperatures, additional calcium release through the sensitized channel feeds

forward in the calcium-ROS/RNS-channel modification cycle.81 Over time, the

chronic elevation in calcium and ROS in YS muscle results in damage to the

mitochondria.
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Mitochondria, in close apposition to triads, are a clear target for a persistent

elevation in local calcium caused by the YS mutation. As a result, mitochondria

could potentially accumulate more calcium than in WT, altering respiration and other

calcium-sensitive processes. This could give rise to the alterations in mitochondrial

membrane potential I observe between WT and YS FDB fibers (compare Figure 1.3B

with Figure 4.1B). In addition, the greater mitochondrial calcium uptake in YS

muscle could increase mitochondrial ROS generation by a variety of mechanisms,

including stimulation of respiration and compromising mitochondrial antioxidant

activity.34 Further, calcium could directly stimulate neuronal nitric oxide synthase

(nNOS), generating NO·, which would indirectly increase ROS by inhibiting complex

IV, and could generate RNS by reacting with ROS. As a result, elevated ROS and

RNS levels in heterozygous tissue could contribute to RyR1 modification and directly

damage mitochondria. Though a certain level of ROS production is expected in

healthy WT muscle, it is anticipated that the YS mitochondria will exhibit a higher

rate of ROS generation. Specifically, I expect increased sites and frequency of

mitochondrial of superoxide production in YS muscle compared to that of WT muscle

may lead to direct mitochondrial damage. Interestingly, Durham et al. detected

increased mitochondrial lipid peroxidation in YS muscle that progressed with age.81

Damage to mitochondrial membranes would certainly alter the metabolic capacity of

mitochondria in YS muscle and, thus, trigger downstream effects on ATP-dependent

processes such as contraction and SR calcium reuptake via SERCA. To maintain the

functional integrity of other mitochondria and preserve gross muscle function, the
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impaired mitochondria would self-segregate from other mitochondria. This would

give rise to the focal regions of damaged mitochondria seen in electron micrographs

and the network superoxide flash detected with the MT-cpYFP imaging (Figure

4.4C). Finally, cores of damaged mitochondria could form as a result of irreparable

mitochondrial damage induced by elevated calcium, ROS, and RNS levels.

However, elevated resting myoplasmic calcium is a characteristic unique to

RyR1 mutations that result in MH/CCD coincidence. Mutations that result in CCD

alone, as discussed previously, have the opposite effect on RyR1, namely inhibiting

calcium permeation. Yet, in both cases, central cores are a common feature. This

suggests that the causative factor in core development may be any type of disruption

in calcium homeostasis, rather than the absolute calcium levels of the myoplasmic

and SR stores. Possibly, as a result of CCD-only mutations in RyR1, there is an

absence of calcium-stimulated mitochondrial respiration during muscle activity,

resulting in a decrease in ROS production. In this case, the decrease in ROS would

trigger a different complement of changes than in YS muscle. Nonetheless, an

imbalance of calcium, as well as ROS and ATP production are likely to lead to

altered SR-mitochondrial signaling, ultimately leading to the formation of central

cores.
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Figure 4.1 JC-1 Emission Ratio is Altered During Postnatal Development in Y522S

Knock-In Mice

A) JC-1 confocal image overlay (J-aggregate [red] and JC-1 monomer [green]

fluorescence, left) and false-colour JC-1 ratio images (J-aggregate/JC-1 monomer, right)

in representative FDB fibers obtained from 0.5, 1, 2, and 4 month-old heterozygous

Y522S knock-in mice. B) Mean (± SE) JC-1 ratio spatial profiles of FDB fibers obtained

from 0.5 (filled circles), 1 (open circles), 2 (filled triangles), and 4 (open triangles)

month-old mice. 1.05 ± 0.02, n = 26 (0.5 m); 1.20 ± 0.02, n = 30 (1 m); 1.37 ± 0.01, n

= 31 (2 m); 0.84 ± 0.01, n = 30 (4 m) at 50% fiber width. *p < 0.01 compared with 4

months at 50% fiber width. Bars: 5 µm.

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Figure 4.2. Mitochondrial Disposition and Sarcomere Spacing in Adult FDB Fibers

A) Confocal images of single adult (8 months-old) FDB fibers from obtained from

wild-type (WT, upper) and heterozygous Y522S knock-in (YS, lower) mice stained

for nuclei (blue, 2.5 µg/ml Hoechst 34580) and mitochondria (red, 10 nM TMRE).

B) TMRE fluorescence along the lines of interest marked in (A). Arrow heads denote

example peak-to-peak distance measurements. C) Box plot of TMRE fluorescence

peak-to-peak distance measurements. Circles, outlying values; left whisker, 10th

percentile; left box boundary, 25th percentile; solid line, median; dotted line, mean;

right box boundary, 75th percentile; right whisker, 90th percentile. Break in X-axis

from 3 to 8 microns. 1.89 ± 0.02 µm, n = 3442 measurements (WT); and 1.87 ± 0.01

µm, n = 2565 (YS).

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Figure 4.3. Mitochondrial Ultrastructure is Altered in FDB Fibers of Y522S

Knock-In Mice

Representative electron micrographs (longitudinal sections) of mitochondria in FDB

fibers obtained from 2 month-old wild-type (WT, A) and heterozygous Y522S knock-

in (YS, B) mice. A) Panels 1-4: Typical WT mitochondria are regularly shaped with

dense matrices and lamellar cristae. B) Panel 1: Regularly shaped mitochondria are

present in YS fibers. Swollen mitochondria with damaged cristae, disrupted external

membranes (filled arrows), and vacuoles (filled stars) are also readily observed.
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Figure 4.4. Mitochondrial Superoxide Flashes in FDB Fibers

A) Representative confocal image of a wild-type (WT) FDB fiber expressing MT-

cpYFP (left) that has been costained for mitochondria with MitoTracker® Red

CMXRos (MTR, middle). Yellow signal indicates colocalization of MT-cpYFP and

MTR fluorescence (right). B and C) Confocal images of peak MT-cpYFP

fluorescence during a superoxide flash (left) and temporal profiles (right) of

superoxide flashes in WT (B) and heterozygous Y522S (YS) knock-in (B) FDB

fibers. Bars: B inset, 1 µm; C inset, 2 µm.

 131

CONCLUSIONS
 132

The close apposition of mitochondria and CRUs in adult skeletal muscle has

been cited as the foundation for numerous physiological processes including

mitochondrial calcium uptake203,212 and suppression of local calcium release by

mitochondria.117,118 Yet, the genesis of the close structural association between SR

and mitochondria has not been investigated in any great detail. Accordingly, this

study characterizes mitochondrial subcellular localization throughout postnatal

skeletal muscle development in fast-twitch glycolytic FDB muscle fibers. Results

indicate a progressive targeting of mitochondria to CRUs. Similar to the irregular

orientation of the sarcotubular network early in skeletal muscle development, the

majority of mitochondria in young FDB muscle (0.5-1 month) are randomly

positioned in longitudinal clusters beneath the sarcolemma and between myofibril

bundles. During the first 4 months of postnatal development, there is a coordinated

increase in CRU density, the number of mitochondria, and mitochondrion-CRU

pairing at the I-band. The rearrangement of mitochondria to CRUs in the fast-twitch

FDB is accompanied by the disappearance of the intermyofibrillar and

subsarcolemma clusters. Additionally, mitochondria in young fibers exhibit a more

negative membrane potential than in adult FDB fibers, suggesting that mitochondrial

function is altered during postnatal development. Finally, mitochondria in adult

FDBs are exclusively positioned adjacent to CRUs on either side of the Z-line.

In their location adjacent to CRUs at the A-I band junction, mitochondria are

aptly positioned for sequestering calcium release from the SR during EC coupling.

Indeed, this work clearly demonstrates that triadic mitochondria in FDB fibers
 133

accumulate calcium during tetanic stimulation, regardless of the stage in postnatal

development (see Chapter 2). Likely, this reflects a through-space coupling rather

than direct “calcium tunneling”212 because mitochondria are positioned on the side of

the SR terminal cisternae opposite to RyR1 feet (see Chapter 1). We have reported

the average distance between RyR1 feet and the outer mitochondrial membrane

(OMM) of an adjacent mitochondrion to be ~130 nm.29 Importantly, this distance is

close to the effective working distance for the slow calcium chelator, EGTA.179

Therefore, the tendency for EGTA to reduce mitochondrial calcium uptake observed

in adult FDB fibers is not surprising. Additional experiments may reveal significance

of the trend indicating that mitochondria accumulate calcium in response to global

elevations, rather than high calcium microdomains. In support of this conclusion,

mitochondrial calcium uptake was not observed during a single twitch. Moreover,

demonstration that longitudinally clustered mitochondria accumulate calcium

following SR calcium release calls into question the source of mitochondrial calcium

uptake and whether close SR-mitochondrial juxtaposition is essential for the process.

In short, determining the conditions under which mitochondria take up calcium and

the dependence upon mitochondrial position with respect to triads will have important

implications for understanding mitochondrial calcium uptake mechanisms and the

presumed privileged SR-mitochondrial calcium signaling.

Furthermore, the importance of SR-mitochondrial calcium uptake is still a

matter of controversy. In particular, does mitochondrial calcium uptake mediate

excitation-metabolism coupling? Theoretically, mitochondrial calcium uptake during

 134

EC coupling may act as a signal to an impending increased demand for ATP.

Calcium activation of matrix dehydrogenases and the F1F0-ATPase would augment

ATP production, and thus, lessen the energy deficit during times of vigorous muscle

activity. Yet, previous studies have shown that the magnitude of mitochondrial

calcium uptake in fast-twitch glycolytic muscle is not sufficient to affect muscle

relaxation or development of fatigue.207 Certainly, though it has not been directly

tested in intact skeletal muscle, excitation-metabolism coupling may be more

important in fast-twitch oxidative/glycolytic and slow-twitch skeletal muscle that

both rely heavily upon oxidative phosphorylation. Therefore, future studies require

measuring NADH levels, ATP production, and mitochondrial calcium uptake

simultaneously and in real time during muscle contraction to determine the

physiological relevance of orthograde excitation-metabolism coupling.

Similarly, defining the role of mitochondria in retrograde suppression of local

calcium release necessitates additional study. The present work demonstrates a

strong correlation between mitochondrial localization and calcium spark suppression

in FDB muscle. Specifically, calcium spark frequency decreases during postnatal

development, concurrent with an increase in mitochondrion-CRU pairing, consistent

with mitochondrial inhibition of local SR calcium release. Further, the data provides

clues into the possible mechanism of calcium spark unmasking by osmotic shock,

namely the disruption of caveolae may trigger downstream redox signaling events.

Consequently, osmotic shock could prove to be a useful method for the study of local

redox control at the CRU and retrograde mitochondria-SR communication.

 135

To conclude, bidirectional retrograde and orthograde SR-mitochondrial

signaling is likely important for calcium homeostasis and control of cellular

metabolism in skeletal muscle. Defects in this local control mechanism could

potentially lead to excessive mitochondrial ROS production that disrupts the cellular

redox state in a manner that contributes to altered Ca2+ release/reuptake. For

example, disruption in SR calcium release due to mutations in RyR1, as well as

muscle cores lacking mitochondrial enzyme activity, are characteristic of the skeletal

muscle disorder Central Core Disease (CCD). However, the mechanistic link

between the calcium dysregulation and mitochondrial pathology remains poorly

understood. Therefore, the final section of the present work began by characterizing

mitochondrial localization, as the foundation for local SR-mitochondrial signaling, in

mouse model of CCD. Although mitochondria in FDB fibers obtained from

heterozygous mice follow the same progressive CRU-targeting as wild-type muscle,

there are significant areas of swollen and damaged mitochondria. The focal nature of

mitochondrial damage in heterozygous muscle fibers implies a breakdown in local

ionic and metabolic homeostasis, likely triggered by the aberrant calcium release (or

leak) from mutant release channels. Clearly, defining the precise mechanism by

which aberrant calcium release initiates pathological changes in mitochondria

demands further investigation and will provide important information regarding the

pathogenesis and potential treatment of CCD. Furthermore, this mechanism by which

changes in muscle ultrastructure, calcium dysregulation, deficits in ATP production,

and oxidative stress disrupt SR-mitochondrial crosstalk is likely common to other

 136

muscle diseases and muscle dysfunction caused by fatigue or exercise-induced

muscle damage. More importantly, calcium dysregulation and an imbalance of ROS

production/scavenging are common facets of cardiac ischemia-reperfusion injury and

neuronal excitotoxicity.34 Therefore, understanding the pathophysiological calcium

and ROS/RNS signaling between SR and mitochondria in skeletal muscle may

provide insight into the analogous processes in other excitable tissues.

 137

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