THE ROLE OF DIFFUSION IN GOVERNING MUSCLE FIBER STRUCTURE AND FUNCTION Ana ...

179 Pages·2011·1.66 MB·English

THE ROLE OF DIFFUSION IN GOVERNING MUSCLE FIBER STRUCTURE AND
FUNCTION

Ana Gabriela Jimenez

A Dissertation Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Doctor of Philosophy

Department of Biology and Marine Biology

University of North Carolina Wilmington

2011

Approved by:

Advisory Committee

Richard Dillaman Bruce Locke

Richard Satterlie Robert Roer

Stephen Kinsey
Chair

Accepted by

Dean, Graduate School
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................... iv
DEDICATION .............................................................................................................................. vii
LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
CHAPTER 1- THE INFLUENCE OF OXYGEN AND HIGH ENERGY PHOSPHATE
DIFFUSION ON METABOLIC SCALING IN THREE SPECIES OF TAIL-FLIPPING
CRUSTACEANS .............................................................................................................................1

Abstract ..........................................................................................................................2
Introduction ....................................................................................................................3
Materials and methods ...................................................................................................7
Results ..........................................................................................................................19
Discussion ....................................................................................................................36
References ....................................................................................................................43
CHAPTER 2- AN EVALUATION OF MUSCLE MAINTENANCE COSTS DURING FIBER
HYPERTROPHY IN THE LOBSTER, HOMARUS AMERICANUS: ARE LARGER MUSCLE
FIBERS CHEAPER TO MAINTAIN? .........................................................................................51

Abstract ........................................................................................................................52
Introduction ..................................................................................................................53
Materials and methods .................................................................................................56
Results ..........................................................................................................................65
Discussion ....................................................................................................................74
References ....................................................................................................................80
CHAPTER 3- BIG AND CHEAP: WHY DO MUSCLE FIBERS GROW SO LARGE? ...........88
Abstract ........................................................................................................................89
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Introduction ..................................................................................................................90
Materials and methods .................................................................................................93
Results ..........................................................................................................................95
Discussion ....................................................................................................................95
References ..................................................................................................................103
CHAPTER 4-NUCLEAR DNA CONTENT VARIATION ASSOCIATED WITH MUSCLE
FIBER HYPERTROPHIC GROWTH IN DECAPOD CRUSTACEANS .................................108

Abstract ......................................................................................................................109
Introduction ................................................................................................................110
Materials and methods ...............................................................................................113
Results ........................................................................................................................121
Discussion ..................................................................................................................129
References ..................................................................................................................133
CHAPTER 5- NUCLEAR DNA CONTENT VARIATION ASSOCIATED WITH MUSCLE
FIBER HYPERTROPHIC GROWTH IN TELEOST FISH .......................................................140

Abstract ......................................................................................................................141
Introduction ................................................................................................................142
Materials and methods ...............................................................................................145
Results ........................................................................................................................152
Discussion ..................................................................................................................158
References ..................................................................................................................162
iii
ACKNOWLEDGMENTS
First and foremost, I would like to acknowledge my committee members, Drs. Steve
Kinsey, Richard Dillaman, Richard Satterlie, Bob Roer and Bruce Locke, thank you all for
providing an enormous amount of support and encouragement during the last five years. I would
like to especially thank my advisor, Dr. Steve Kinsey, for always being a patient and supportive
role model, for never losing faith in me or in my capacity to do science, but most importantly I
would like to thank Steve for challenging my limits and forcing me to surpass them, and for
making me a good scientist and giving me a strong foundation from which to launch from. I will
forever be indebted to you, Steve, for all the time and effort you spent in making me the scientist
I am today, thank you from the bottom of my heart. Dr. Dillaman, your laugh and enthusiasm
for all things science-y is contagious and put many smiles on my face throughout the years; Dr.
Satterlie and Dr. Roer, thank you for teaching me topics outside my immediate realm of comfort
and for your patience; Dr. Locke, thank you for teaching me your engineering language and
providing me with knowledge to understand fields other than my own. I would also like to thank
someone I consider an “unofficial” committee member, Mark Gay, for all of his help with all
things microscopy or image related, for always having a solution to any problem I ever brought
down to him and for being a great friend. Dr. Fritz Kapraun played an essential role in
incorporating some new concepts to our lab and also within my dissertation, I would like to
thank him for all of the time and effort he devoted to my last two chapters. Dr. Pabst, who has
been an incredible source of support as a graduate coordinator and fellow scientist and whose
advice never let me stray, thank you for inspiring me to love science as much as you do.
Secondly, I could not have done any of this work without many individuals who helped
with the collection of all the animals used in this research. Some of these people include: Captain
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Jeff Wolfe who collected many fishes and crustaceans for me, without Jeff‟s happy demeanor
and complete selflessness, a lot of these animals could not have been collected; Captains Jay
Styron, Ken Johns, and Gerry Compeau for allowing me to tag along in many trawling trips and
for helping me keep specimens alive; Jeremy Burnett for allowing me to get on the R/V Cape
Fear while his summer camps were going on; My friends who came out with me on repeated
occasions to collect: Leigh Anne Harden, Kenneth Kelly, Carolina Priester, Trent Ross, Jeff
Harms, Daniel Russo, Buddy Trost, Ginger Winder, and Jessica Snoddy; Dr. T. Lankford, and
Ed Arb for all the seining help. I would also like to especially thank my advisor‟s kids, Ellie and
Nathan Kinsey, who also selflessly gave up after school hours to help me seine, dip net, and
transport large amounts of animals, and for also providing comic relief throughout the years.
Thirdly, I would like to thank my friends and lab mates. Jessica Snoddy, Will
Snoberger, Stacy Galleher and Daniel Russo, your friendship has meant the world to me, thank
you for keeping me around in the good and the bad times, for showing me what true friendship is
all about, and for allowing me to trust you guys with everything that goes on in my life. Jeff
Overton, Al Nyack, Trent Ross and Kenneth Kelly, you all made the lab my home, and a fun and
interactive place to do science; you challenged me intellectually at all times, you provided heated
and non heated arguments about science and through this process we grew and developed as
scientists together, for this, I thank you.
Last, but most definitely not least, I have to acknowledge and thank my family who has
been my rock and my pushing force through this whole process. Words cannot express how
thankful I am to each and every one of these people. Mom, thank you for always keeping me
afloat when I would have for sure sank, thank you for all of your love and dedication to us. Dad,
thanks for your words of wisdom when I needed them and thanks for supporting my dream. To
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my sister, Daniela, your mere presence in my life brings a constant smile to my face, you make
this world a better place and for that I thank you. To my grandmother, even though you
constantly worry about me being eaten by a shark one day, thank you for always being so proud
of your granddaughter. To my cousin, Tatiana, thank you for growing up beside me as a sister.
To all of my family in Venezuela, the distance may keep us apart physically, but I always know
you are right here with me, thank you for your support and love always. To my grandfather, you
were the first one I sent all of manuscripts to and one of the only people whose opinion mattered
to me, thank you for all of your input, inquiries, encouragement and most importantly for always
reminding me to strive to be a better person and scientist.
Funding agencies that supported this work include the National Science Foundation,
National Institute of Arthritis and Musculoskeletal and Skin Diseases, Sigma Xi and Society for
integrative and comparative biology (SICB) grants-in-aid programs. As well as UNCW‟s
department of Biology and Marine Biology, Biology Graduate Student Association, the Graduate
School and the Graduate Student Association.

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DEDICATION
I would like to dedicate this manuscript to my grandfather, Luis Jiménez Segura, who
was an incredible, admirable human being, a fantastic role model, and a great man who wore all
of his “life hats” with incredible grace and ease. My love for the ocean started when, as a child,
he would take me on walks along the beach and tell me stories about the ocean and its critters.
He is the sole culprit for my love of all things marine. But most importantly, he is the man that
never lost faith that I would make something great happen
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LIST OF TABLES
CHAPTER 1 Page
1. Parameters used in reaction-diffusion model ..............................................................34
2. Percentage of VO devoted to AP re-synthesis in muscle ...........................................41
2
CHAPTER 3
1. Species information, body mass and fiber diameter of each size class .......................96
2. Rates of depletion of arginine phosphate (AP) or phosphocreatine (PCr) to measure
cost in the absence of Na+-K+-ATPase ATP demand ......................................................101

CHAPTER 4
1. Diploid (2C) nuclear DNA content in hemocytes of eight species of decapod
crustaceans ......................................................................................................................124

2. Hypertrophic growth in white muscle fibers of eight species of decapod
crustaceans .......................................................................................................................125

CHAPTER 5
1. Diploid (2C) nuclear DNA content in erythrocytes of seventeen species of fishes .. 146
2. Hypertrophic growth in white muscle fibers of seventeen species of fishes .............153
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LIST OF FIGURES
CHAPTER 1 Page
1. Schematic of mathematical model showing one dimensional spatial domain ............14
2. Standard (A) and post-exercise (B) VO for all three species and size classes ...........22
2
3. Abdominal white muscle cross-sectional fiber diameter distribution and mean
diameter (inset) from each size class of the three species of crustaceans ..........................23

4. Scaling with body mass of citrate synthase activity from abdominal white muscle ...24
5. The initial rate of post-tail flip arginine phosphate recovery for the small and large
size classes .........................................................................................................................26

6. Scaling with body mass of the AP/PCr recovery rate in white muscle from
crustaceans, fishes, mollusks and the crustaceans from our current study .......................27

7. Comparison of the relationship between the scaling with body mass of (A) whole
animal metabolic rate (VO ) and the scaling of a metabolic process (AP re-synthesis)
2
following tail- flipping responses in three species of crustaceans, and (B) muscle aerobic
capacity (CS activity) and AP recovery rate .....................................................................28

8. Lactate levels immediately after exercise (black bars) and after 15 min of recovery
(gray bars), as well as the relationship between contractile lactate production and number
of tail flips ..........................................................................................................................29

9. Mathematical model of reaction rate for the case without diffusion as a function of the
two reaction rate constants with a fixed diffusion distance (L=104.5 µm) and O
2
boundary concentration of 7.85 M ..................................................................................30

10. Mathematical model of reaction rate and effectiveness factor for the case with
diffusion for various values of k and k with a fixed diffusion distance (L=104.5) and O
1 2 2
boundary concentration of 7.85 M ..................................................................................31

11. Examples of reaction-diffusion model output for the smallest and largest fibers .....33
12. Effectiveness factor as functions of rate and length compared to experimental
data .................................................................................................................................... 35

CHAPTER 2
1. Abdominal white muscle fiber diameter distribution, mean diameter (top inset) and
SA:V (bottom inset) from small and large size classes of H. americanus ........................68
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2. Example of 31P-NMR spectra where (A) is an initial spectrum from an adult lobster in
the treatment containing cyanide and iodoacetate, (B) is the final spectrum after 100 min
in the presence of cyanide and iodoacetate, and (C) is a spectrum after 100 min in
oxygenated saline in the absence of inhibitors ..................................................................69

3. The rate of arginine phosphate (AP) depletion for the small (top) and large (bottom)
size classes used to measure metabolic costs .....................................................................70

4. (A) Mean AP depletion rate for each treatment in small and large size classes of H.
americanus. (B) Amount of ATP consumed per minute for each process ........................71

5. Metabolic capacity and comparison to metabolic cost in anaerobic fibers of small and
large size classes of H. americanus. (A) Maximal Na+-K+-ATPase activity. (B) Maximal
SR-Ca2+- ATPase activity. (C) Nuclear number volume. (D) Fractional change in
measurements of capacity and cost during growth from the small to large size class.......72

6. (A) Immunolocalization of the Na+-K+-ATPase revealed that Na+-K+-ATPase pumps
were largely localized to the sarcolemma in H. americanus abdominal muscle fibers
(N=3). (B) Cross-section of white abdominal muscle fibers from wheat germ agglutinin
Alexa Fluor 488 (WGA) injected adults lobsters ..............................................................73

7. Relationship between the Na+-K+-ATPase cost and diffusion limitation of aerobic
metabolism during hypertrophic fiber growth in H. americanus. .................................... 79

CHAPTER 3
1. Example of a set of NMR spectra (A) and AP depletion data under the two treatment
conditions for juvenile P. argus ........................................................................................ 97

2. Relative changes in NA+-K+ ATPase cost (A) and maximal enzymatic activity (B)
during hypertrophic fiber growth for all species................................................................98

3. Comparisons between crustacean and fish Na+-K+ ATPase cost (A), Na+-K+ ATPase
activity (B), muscle basal metabolic cost (C), and relative Na+-K+-ATPase cost (D) ....102

CHAPTER 4
1. Cross-section of Homarus americanus anaerobic abdominal muscle fiber from adult
animal injected with wheat germ agglutinin Alexa Fluor 594 (WGA). further treated with
DAPI, a fluorescent probe for nuclei ...............................................................................117

2. Relationship between nuclear area and 2C DNA content correlated from adult
hemocytes in eight species of decapod crustaceans .........................................................118

3. Histogram of nuclear DNA content from anaerobic muscle fibers of juvenile and
adult specimens of eight species of decapod crustaceans ................................................126
x