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SKELETAL MUSCLE ADAPTATIONS
IN CACHECTIC, TUMOR-BEARING RATS
Dissertation
Jeffrey S. Otis, Ph.D.
April 4, 2003
Department of Human Nutrition, Foods and Exercise
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061
Committee Members
Jay H. Williams, chair
Robert W. Grange
William R. Huckle
David Moore
John Robertson
Key Words: Skeletal Muscle, Cancer Cachexia, Calcineurin, Myosin Heavy Chain
ABSTRACT
Skeletal muscle adaptations in cachectic, tumor-bearing rats
Jeffrey S. Otis, Ph.D.
Cancer cachexia is a debilitating, paraneoplastic syndrome commonly associated with
late stage malignancy. It is estimated that ~25% of cancer-related deaths are due directly
to complications arising from cachexia (Barton, 2001). Cachexia manifests as severe
body wasting, primarily due to the loss of skeletal muscle mass.
This study tested the hypothesis that muscle atrophy associated with cancer cachexia
could be attenuated by using a unilateral, functional overload (FO) model applied
concurrently with tumor development. To accomplish this, Morris hepatoma MH-7777
cells were implanted in adult female, Buffalo rats (n = 12) and allowed to incubate for 6
weeks. FO surgeries (n = 12) were performed five days prior to MH-7777 cell
implantation.
Over the course of six weeks, healthy, age, sex and strain-matched, vehicle- injected rats
(n = 12) gained ~5% of body weight compared to tumor-bearing rats that lost ~6% of
body weight when adjusted for tumor mass. Tumor-bearing animals experienced
significant atrophy to gastrocnemius, tibialis anterior, extensor digitorum longus,
plantaris and diaphragm muscles.
FO successfully reversed plantaris muscle atrophy in cachectic, tumor-bearing rats (n=5).
FO plantaris masses were ~24% larger than contralateral controls. However, this
hypertrophic response was not as great as FO plantaris muscles from healthy, sham-
operated controls (~44% larger than contralateral controls, n=5). FO plantaris muscles
from tumor-bearing rats had ~1.5 fold increase in myonuclei/fiber ratios compared those
of sham-operated, tumor-bearing controls (n = 6). Therefore, cancer cachexia did not
prevent myonuclear accretion necessary for skeletal muscle hypertrophy.
iii
Little data exists on adaptations to myosin heavy chain (MHC) isoforms in cachectic
skeletal muscle. Plantaris muscles from tumor-bearing rats displayed decreased
percentages of MHC type I compared to plantaris muscles from vehicle- injected controls
(7% vs. 3%, respectively). However, FO plantaris muscles from tumor-bearing rats had
an increased percentage of MHC type I and decreased percentage of MHC type IIb
compared to sham-operated tumor-bearing rats, adaptations commonly seen in trained
muscles. Therefore, cancer cachexia did not prevent the capability of skeletal muscle to
respond normally to hypertrophic stimuli.
This study also attempted to characterize a mechanism responsible for the hypertrophic
response, increased myonuclei/fiber ratio and transition toward a slower MHC profile in
FO plantaris muscles from tumor-bearing rats. Recently, the Ca2+/calmodulin-dependent
protein phosphatase, calcineurin, has been suggested as a critical factor regulating
skeletal muscle growth and fiber-type dependent gene expression (Chin, 1998; Wu, 2000;
Olson, 2000; Otis, 2001). The protein content of the catalytic subunit (CaNα) and the
regulatory subunit (CaNβ) of calcineurin were unchanged in plantaris muscles from
tumor-bearing animals compared to healthy controls. Furthermore, total and specific
(normalized to CaNα protein content) calcineurin phosphatase activity were not altered in
any group. Therefore, calcineurin activity did not appear to be associated with the
regulation of the morphological and physiological response of hypertrophying plantaris
muscles in cachectic, tumor-bearing rats.
Overall, this study indicated that atrophied plantaris muscles from tumor-bearing animals
have a reduced capacity to hypertrophy potentially due to a decreased myonuclei/fiber
ratio. Furthermore, it is unlikely that changes to mass and MHC isoform expression are
associated with calcineurin phosphatase activity.
iv
ACKNOWLEDGEMENTS
I must thank my committee members: Jay Williams (chair), Rob Grange, Bill Huckle,
David Moore and John Robertson for their patience and guidance.
The support and expertise that I have received from colleagues and lab technicians has
been extremely valuable. Special thanks are extended to Simon Lees, Tod Distler, Janet
Rinehart and Kathy Reynolds.
However, I do not thank my family – Dad, Mom, Arlene, Debra, Stevie and Jimmy –
because thanking them would be the same thing as thanking myself.
v
TABLE OF CONTENTS
ABSTRACT........................................................................................................................ ii
ACKNOWLEDGEMENTS ................................................................................................iv
TABLE OF CONTENTS.....................................................................................................v
LIST OF FIGURES ......................................................................................................... viii
LIST OF TABLES ..............................................................................................................ix
TABLE OF ABBREVIATIONS .........................................................................................x
CHAPTER ONE – INTRODUCTION............................................................................... 1
Statement of Problem...................................................................................................... 2
Significance of Study...................................................................................................... 3
Specific Aims .................................................................................................................. 4
Specific Aim 1 ............................................................................................................ 5 Rationale ................................................................................................................. 5 Hypothesis............................................................................................................... 6
Specific Aim 2 ............................................................................................................ 6 Rationale ................................................................................................................. 6 Hypothesis............................................................................................................... 6
Specific Aim 3 ............................................................................................................ 7 Rationale ................................................................................................................. 7 Hypothesis............................................................................................................... 7
CHAPTER TWO – REVIEW OF LITERATURE............................................................. 8
Cancer Cachexia ............................................................................................................. 9
Cancer Cachexia: Energy expenditure of tumors ........................................................... 9
Cancer Cachexia: Effect on Host Metabolism.............................................................. 10
Cancer Cachexia: Effect on Skeletal Muscle Protein Turnover ................................... 13
Cancer Cachexia: Effect on Skeletal Muscle Physiology............................................. 14
Calcineurin .................................................................................................................... 18
Skeletal Muscle Contraction and Calcineurin Signaling .............................................. 21
Calcineurin: Role in Skeletal Muscle Protein Expression and Hypertrophy................ 24
Role of Myonuclear Apoptosis in Skeletal Muscle Atrophy ........................................ 28
Role of Myonuclear Accretion in Skeletal Muscle Hypertrophy ................................. 29
Summary....................................................................................................................... 31
CHAPTER THREE - METHODS .................................................................................... 32
vi
Study I: Morphological changes to skeletal muscles from tumor-bearing rats ............ 33
Study II: Effects of hypertrophic stimulus on cancer cachexia .................................... 33
Animals ......................................................................................................................... 33
Tumor Model ................................................................................................................ 34
Ablation Surgeries......................................................................................................... 34
Muscle Preparation ....................................................................................................... 35
ATP-independent proteasome activity assay................................................................ 35
Western Blotting for CaNα and CaNβ ......................................................................... 36
Calcineurin Phosphatase Activity Assay ...................................................................... 37
Electrophoretic Separation of MHC isoforms .............................................................. 38
Determination of Myonuclear Density ......................................................................... 38
Statistics ........................................................................................................................ 39
Limitations .................................................................................................................... 39
Basic Assumptions ........................................................................................................ 40
CHAPTER FOUR - RESULTS ........................................................................................ 41
Study I: Morphological Changes to skeletal muscles from tumor-bearing rats............ 42
Study I: Tumor Masses ............................................................................................. 42 Study I: Body Masses ............................................................................................... 42 Study I: Skeletal Muscle Masses .............................................................................. 43
Study II: Effects of hypertrophic stimulus on cancer cachexia .................................... 44
Study II: Body Masses .............................................................................................. 44 Study II: Plantaris Muscle Masses ............................................................................ 44 Study II: Myosin Heavy Chain (MHC) Isoform Expression.................................... 45
MHC type I ........................................................................................................... 46 MHC type IIa ........................................................................................................ 46 MHC type IIx........................................................................................................ 46 MHC type IIb ........................................................................................................ 47
Study II: Myonuclei/Fiber Ratios ............................................................................. 47 Study II: Calcineurin Subunit Expressions ............................................................... 48
Calcineurin-α (CaNα) expression ........................................................................ 48 Calcineurin-β (CaNβ) expression......................................................................... 49
Study II: Calcineurin Phosphatase Activities ........................................................... 49 CHAPTER FIVE - DISCUSSION.................................................................................... 65
Introduction................................................................................................................... 66
Major Findings .......................................................................................................... 66 Specific Aims ............................................................................................................ 67
Body mass loss and skeletal muscle atrophy associated with cancer cachexia ............ 68
vii
Hypertrophic response of atrophied muscles from cachectic, tumor-bearing rats........ 70
Myosin heavy chain isoform composition.................................................................... 71
Influence of myonuclear apoptosis and accretion......................................................... 73
Calcineurin activity is not associated with plantaris hypertrophy ................................ 74
Conclusions ................................................................................................................... 76
Future Directions ........................................................................................................... 77
CHAPTER SIX - REFERENCES .................................................................................... 78
APPENDIX....................................................................................................................... 91
CURRICULUM VITAE................................................................................................... 98
viii
LIST OF FIGURES
FIGURE 1. Metabolic interactions between tumor and host....................................... 12
FIGURE 2. Calcineurin domain structures .................................................................. 19
FIGURE 3. Calcineurin-mediated signaling pathway ................................................. 21
FIGURE 4. Overview of muscle contraction............................................................... 23
FIGURE 5. Immunohistochemical staining of MCK-CN* plantaris fibers ................ 25
FIGURE 6. CaN-dependent and CaN-independent stages during regrowth ............... 30
FIGURE 7. Correlation between tumor mass and percent change
in adjusted body mass .............................................................................. 51
FIGURE 8. Eating habits of healthy and tumor-bearing rats over 6 weeks ................ 52
FIGURE 9. Plantar flexor masses from tumor-bearing rats over time ........................ 53
FIGURE 10. Dorsi flexor and diaphragm masses from tumor-bearing rats over time 54
FIGURE 11. Absolute changes to body mass over 6 weeks of tumor development ... 56
FIGURE 12. Percent changes from initial body mass over 6 weeks ........................... 57
FIGURE 13. Plantaris mass increases after functional overload ................................. 59
FIGURE 14. Myosin heavy chain isoform distribution............................................... 60
FIGURE 15. Myonuclear density ................................................................................ 61
FIGURE 16. Calcineurin-alpha subunit expression..................................................... 62
FIGURE 17. Calcineurin-beta subunit expression....................................................... 63
FIGURE 18. Specific calcineurin phosphatase activity............................................... 64
FIGURE 19. Raw tracing from ATP-independent
ubiquitin, proteasome activity assay...................................................... 94
FIGURE 20. Plantaris stained for dystrophin and counterstained for myonuclei ....... 95
FIGURE 21. Morris Hepatoma .................................................................................... 96
FIGURE 22. ATP-dependent-ubiquitin proteasome system ....................................... 97
ix
LIST OF TABLES
TABLE 1. Differences between MHC fiber types in human skeletal muscle ............. 15
TABLE 2. Body masses form tumor-bearing rats
at 2, 4 or 6 weeks of tumor growth........................................................... 50
TABLE 3. Maximal proteasome activity..................................................................... 55
TABLE 4. Absolute plantaris muscle masses .............................................................. 58
TABLE 5. Morphological data .................................................................................... 92
TABLE 6. MHC, myonuclei/fiber ratios, calcineurin subunits and activities data…..93
x
TABLE OF ABBREVIATIONS
Ach Acetylcholine Akt/mTOR Akt/mammalian target of rapamycin AI Autoinhibitory AMC 7-amino, 4-methyl coumarin AP-1 Activator protein-1 ATP Adenosine triphosphate AU Arbitrary Units bHLH Basic Helix-Loop-Helix [Ca2+]i Intracellular calcium concentration CaN Calcineurin CHF Congestive heart failure CM Calmodulin CsA Cyclosporin A DHPR Dihydropyridine receptor EDL Extensor digitorum longus EF EF hand motif EPA Eicosapentaenoic acid FK506 Tacrolimus FKBP FK binding protein FO Functional overload Gastroc Gastrocnemius GSK-3ß Glycogen synthase kinase-3ß IFN? Interferon-? IGF-1 Insulin- like growth factor-1 IL-1 Interleukin-1 LPL Lipoprotein lipase MCK-CN* Muscle creatine kinase-calcineurin transgene MEF-2 Myocyte enhancing factor-1 MH-7777 Morris hepatoma-7777 MHC Myosin heavy chain MLC-2 Myosin light chain-2 MPC Muscle precursor cell MRF Muscle regulatory factors NFATc1 Nuclear factor of activated T cells-c1 NF?B Nuclear factor ?B NLS Nuclear localization sequence PGAM-M Phosphoglycerate mutase – muscle isoform PIF Proteolysis inducing factor PP Protein phosphatase RAP Rat antibody production test RyR Ryanodine receptor S Sham-operated SERCA Sarcoplasmic, endoplasmic reticulum calcium ATPase
xi
SR Sarcoplasmic reticulum TA Tibialis anterior TB Tumor-bearing TNFa Tumor necrosis factor-a TnI Troponin inhibitory protein VEGF Vascular endothelial growth factor Veh Vehicle- injected
1
CHAPTER ONE – INTRODUCTION
2
Statement of Problem
Cancer cachexia is a debilitating, paraneoplastic syndrome commonly associated with
late stage malignancy. It is estimated that 22-30% of cancer-related deaths are due
directly to complications arising from cachexia (Barton, 2001). Cachexia results in
severe body wasting, primarily due to loss of skeletal muscle mass, as tumor and host
compete for nutrients. Cachectic patients present severe asthenia, generalized weakness
and decreased ability to endure contemporary anti-neoplastic therapies that often result in
decreased survival times (Baracos, 2000; Fearon, 2002).
The complexity of skeletal muscle fiber types (i.e., fast versus slow muscle types), the
relationships between structural integrity and mechanical function and the sensitivity of
skeletal muscle to metabolic perturbations have made successful clinical intervention for
cachectic patients difficult. While strength training and nutritional supplementation
partially attenuate the extent of muscle atrophy, these interventions are often difficult to
prescribe due to the constraints of contemporary chemotherapy including fatigue, anemia
and nausea (Evans, 2002). Preclinical studies in rodents that receive anabolic hormones,
such as growth hormone, growth hormone-releasing hormone, insulin- like growth factor
(IGF-1), and IGF binding protein 3, might reverse the catabolic state associated with
cachexia in cancer patients (Draghia-Akli, 2002). Nonetheless, researchers have
identified key regulators of anabolic pathways and characterized molecular pathways
involved in muscle growth (for review see Glass, 2003). The exploitation of these
molecular regulators and anabolic pathway through gene therapy techniques might open
potential avenues for intervention in cancer cachexia (Rosenthal, 2002).
Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, has recently been
suggested as a critical factor regulating skeletal muscle growth. Calcineurin
dephosphorylates nuclear factor of activated T cells (NFATc1), which exposes the
nuclear localization sequence and allows for NFATc1 nuclear translocation. Nuclear
NFATc1 binds to conserved DNA sequences that activate the transcription of several
growth regulatory genes and fiber type-specific genes (Chin, 1998; Wu, 2000; Olson,
2000). Furthermore, calcineurin has also been suggested to play an important role in
3
myonuclear accretion, a necessity for skeletal muscle growth (Mitchell, 2002).
Therefore, exploiting calcineurin-mediated pathways through gene therapy approaches
might serve as potential therapeutic interventions for skeletal muscle wasting associated
with cancer cachexia.
Significance of Study
Although cancer cachexia results in significant muscle mass loss and generalized
weakness, little is known about its effects on other aspects of muscle function such as
force-velocity characteristics, power output, metabolic properties, myosin heavy chain
(MHC) isoform composition and fatigability. Certainly, other disuse and/or disease
models, including congestive heart failure (CHF) (Spangenburg, 2002), limb
immobilization (Jänkälä, 1997), spinal cord injury (Jiang, 1991; Talmadge, 1995) and
hindlimb suspension (Bigard, 1997) result in muscle atrophy and have well documented
changes to muscle function and protein expression. Our laboratory has indicated that
functional overload (FO) increased plantaris mass and fiber cross-sectional areas in CHF
rats and altered Ca2+-uptake rates and MHC isoform expression to resemble trained
muscles (Spangenburg, 2002). Therefore, strength training may also improve various
aspects of skeletal muscle physiology in cachectic cancer patients. In fact, to the author’s
knowledge, only one study has analyzed changes to MHC isoform composition in tumor-
bearing, cachectic animals (Diffee, 2002). Briefly, the current study has elaborated upon
and clarified the complexity of adaptation to skeletal muscle mass, MHC isoform
composition and myonuclei/fiber ratio that occur secondary to cancer cachexia.
Calcineurin-dependent pathways may potentially regulate skeletal muscle mass (Dunn,
1999; Talmadge 2000a; Spangenburg, 2001), fiber-type specific gene expression (Chin,
1997) and protein expression (Otis, 2001). Although some studies have failed to
implicate calcineurin as a key regulator in these processes (Swoap, 2000; Dupont-
Versteegden, 2002), a potential role for calcineurin-dependent regulation of skeletal
muscle mass and fiber-type specific protein expression in tumor-bearing, cachectic rats is
intriguing.
4
Specific Aims
The FO model was used in the current study to induce skeletal muscle hypertrophy to
identify a potential role for calcineurin in regulating skeletal muscle mass,
myonuclei/fiber ratios and MHC isoform expression in tumor-bearing, cachectic rats.
Functional overload of the plantaris was achieved by gastrocnemius tenotomy. This
eliminates any weight bearing support provided by the gastrocnemius. Generally, the
gastrocnemius comprises ~80% of the cross-sectional area of the plantar flexor group
(Lieber, 2002), however, following ablation of the gastrocnemius tendon, ~20% of the
plantar flexor musculature is available. Therefore, a greater demand (i.e., increased
neuromuscular activity and load bearing requirements) is placed on the synergistic
plantaris and soleus muscles. This model has been used extensively to mimic the effects
of strength training. For instance, after one and ten weeks of FO, rat plantaris muscles
were 22 and 56% greater, respectively, than plantaris muscles from age-matched controls
(Roy, 1997). Our lab indicated ~30% increase in plantaris muscle mass nine weeks after
unilateral FO compared to controls (Spangenburg, 2002). The FO model was used in the
current study to provide a significant hypertrophic stimulus to potentially counteract the
muscle wasting associated with cancer cachexia. Additionally, it was of interest to
determine if cancer cachexia would affect the fast-to-slow MHC isoform transition in FO
plantaris, a common transition evident in trained muscle (Roy, 1997). Moreover, because
calcineurin has been linked to regulating both muscle mass and fiber type-specific gene
expression, potential alterations to calcineurin subunit expression and phosphatase
activity in tumor-bearing, cachectic rats were intriguing.
Preliminary studies from rats bearing a subcutaneous Morris hepatoma for 6 weeks
indicated increased proteasome activity in select muscles (Table 3). Significant atrophy
was evident in extensor digitorum longus (EDL), tibialis anterior (TA), diaphragm,
plantaris and gastrocnemius muscles (Figures 9 and 10). The current study attempted to
attenuate these changes by introducing a significant hypertrophic stimulus concurrent
with tumor development. Accordingly, specific alterations to body mass, plantaris
muscle mass, MHC isoform composition, myonuclei/fiber ratios, calcineurin subunit
protein content and calcineurin enzyme activity will be described.
5
Specific Aim 1
The purpose of the first specific aim was to identify the effects of FO on plantaris muscle
masses and myonuclei/fiber ratios in tumor-bearing rats compared to tumor-bearing,
sham-operated controls.
Rationale
Reduced myonuclear numbers in skeletal muscle have been reported in a variety of
atrophy models, including, spinal cord isolation (Allen, 1995), microgravity (Allen,
1996), chronic denervation (Rodrigues, 1995) and hindlimb suspension (Darr, 1989).
Although no studies to date have analyzed myonuclear number in muscles from tumor-
bearing, cachectic animals, it would be surprising if total myonuclei counts were not
reduced similar to other atrophy models. While the molecular mechanisms behind
reduced myonuclear number during skeletal muscle atrophy are currently not well
defined, it is thought “modified” apoptosis mechanisms cause myonuclear death without
subsequent cell destruction (Allen, 1999).
Skeletal muscle hypertrophy is dependent on myonuclear accretion, the fusion of satellite
cells to the fiber proper to increase the number of myonuclei. FO increases satellite cell
number during the early stages of hypertrophy (~3-4 days) followed by a slight decrease
in satellite cell number to control levels and an increase in total myonuclei number during
the later stages of hypertrophy (~2-4 weeks) (Snow, 1999). The decrease in satellite cell
number during the later stages of hypertrophy represents the incorporation of new
myonuclei into the fiber proper evidenced by the increase in total myonuclei. Blocking
satellite cell proliferation and fusion with radiation treatments successfully prevents the
hypertrophic response normally associated with overloaded muscle (Rosenblatt, 1992;
1994), thus supporting a causal link between satellite cell proliferation, myonuclear
accretion and skeletal muscle hypertrophy.
6
Hypothesis
It was hypothesized that FO would attenuate atrophy and increase the myonuclei/fiber
ratio in plantaris muscles from tumor-bearing rats compared to tumor-bearing, sham
operated controls.
Specific Aim 2
The second specific aim was to identify the effect of FO on MHC isoform composition in
tumor-bearing rats compared to controls.
Rationale
One week after FO surgeries, rat plantaris muscle contains significantly greater
percentages of type I/IIa hybrid fibers (i.e., fibers containing both MHC type I and IIa
isoforms) compared to controls (Ta lmadge, 1997). After 10 weeks, overloaded plantaris
fibers contain 35% MHC type I compared to 10% in normal weight bearing controls. A
fast-to-slow transition of MHC isoforms following FO is also evident in mouse (Awede,
1999) and cat (Talmadge, 1996a) models.
To date, very few studies have reported changes to skeletal muscle MHC isoforms in
tumor-bearing animals. Diffee and colleagues (2002) reported de novo expression of
MHC type IIb in soleus muscles from mice bearing a murine C-26 adenocarcinoma, but
pilot data for the current study demonstrated no change in MHC isoform composition of
the rat soleus muscle after 2, 4 or 6 weeks of tumor development. To the author’s
knowledge, no report has investigated the combined effects of FO and cancer cachexia on
MHC isoform composition.
Hypothesis
It was hypothesized that FO would cause a shift toward slower isoforms of MHC in both
tumor-bearing rats and healthy controls.
7
Specific Aim 3
The third specific aim was to identify the effect of FO on calcineurin subunit protein
content and calcineurin phosphatase activity in tumor-bearing rats compared to controls.
Rationale
Calcineurin is a heterodimer composed of catalytic (CaNa) and regulatory (CaNß)
subunits. CaNa protein levels are higher in the fast-type plantaris muscle compared to
the slow-type soleus (Spangenburg, 2001; Mitchell, 2002). However, CaNß protein
levels in the plantaris muscle are ~59% less than the slow-type soleus (Mitchell, 2002).
Because CaNß is required for functional phosphatase activity, a smaller proportion of
total CaNa should be enzymatically active in the plantaris muscle. Indeed, the plantaris
muscle has been shown to have less calcineurin activity compared to the soleus muscle
(Mitchell, 2002). However, it is unclear if CaNa and CaNß protein content or calcineurin
phosphatase activity would be altered FO plantaris muscles from tumor-bearing,
cachectic rats.
Hypothesis
Because calcineur in activity has been associated with regulation of skeletal muscle mass
and slow fiber- like protein expression, it was hypothesized that atrophied plantaris
muscles from tumor-bearing animals might display reduced subunit protein content
and/or reduced calcineurin activity. It was hypothesized that FO of plantaris muscles
from tumor-bearing rats would cause calcineurin subunit protein content and phosphatase
activity to be comparable to control muscles.
8
CHAPTER TWO – REVIEW OF LITERATURE
9
Cancer Cachexia
Cancer cachexia, also known as wasting syndrome, is a paraneoplastic syndrome
afflicting approximately two thirds of patients who die with advanced cancer (Argiles,
1997). An estimated 22-30% of all cancer deaths are directly attributable to
complications arising from cachexia, including the deterioration of respiratory
musculature and subsequent hypostatic pneumonia (Warren, 1932; Barton, 2001).
Cachexia is a pathological state characterized by severe weight loss, and can be
accompanied by anorexia, asthenia, general weakness and/or anemia. Cachexia has been
associated with many chronic diseases, including cancer, AIDS and emphysema (Barton,
2001). It is generally accepted that the underlying cause of cachexia is invariably a
severe metabolic disturbance as host and tumor compete for nutrients and energy
substrates. The result of this battle over substrates is an increased catabolism of protein
and lipid stores for conversion into energy.
Cancer cachexia appears to be tumor-type dependent. For instance, patients with lung
and upper gastrointestinal cancer are more likely to experience cachexia-induced weight
loss compared to patients with breast and lower gastrointestinal cancer (DeWys, 1980).
However, although certain tumor types are more commonly associated with cachexia,
patients with the same tumor type can vary in extent to which they become cachectic.
Such observations point to tumor phenotype and/or host genotype contributing to the
development of cachexia and emphasize the importance of host-tumor interaction in the
pathogenesis of this syndrome (Monitto, 2001).
Cancer Cachexia: Energy expenditure of tumors
The energy metabolism of cancer cells has been the focus of investigation since it was
suggested 73 years ago that aerobic glycolysis (i.e., lactate production in the presence of
oxygen) was a phenomenon peculiar to tumors (Warburg, 1929). Tumors tend to utilize
very large amounts of glucose to fulfill their high-energy requirements to support protein
synthesis and growth. This accumulation of glucose molecules is primarily accomplished
10
by lactate recycling via gluconeogenesis (i.e., Cori cycle), however glucose is also
produced from the gluconeogenic amino acid, alanine, secondary to skeletal muscle
protein degradation. In addition, small contributions of glucose come from the liberation
of glycerol molecules from adipose tissue (Figure 1).
Like other cells, tumor cells require a blood supply to deliver nutrients and remove
cellular wastes. Most tumor masses larger than 1-mm3 contain regions of low oxygen
tension due to an imbalance of oxygen supply and consumption (Dachs, 2000). Above
this volume, the extent of cellular growth at the periphery is balanced by the necrosis of
the oxygen- and nutrient-deprived cells at the tumor core. This constraint on growth is
overcome by the release of various vascular growth factors from the tumor proper or
from infiltrating immune cells (e.g., Vascular Endothelial Growth Factor, VEGF)
(Senger, 1983). These factors help to stimulate angiogenesis, the formation of new blood
vessels, and improve blood flow to the tumor cells. Indeed, recent cancer therapy
approaches attempt to minimize neovascularization and deprive tumor cells of nutrients
required to support protein synthesis, growth and metastasis (Ghilardi, 2001).
Cancer Cachexia: Effect on Host Metabolism
Patients suffering from cachexia often report decreased appetite or anorexia- like
symptoms (Argiles, 1997). However, in otherwise healthy anorexic patients, the liver
produces ketone bodies to supply carbon skeletons for energy production and spares
protein stores, but this does not occur with cancer cachexia. In cachectic cancer patients,
ketone bodies are not produced and the breakdown of protein stores for energy is
unrestricted. While anorexia or reduced caloric intake may contribute to weight loss
associated with cancer cachexia, they cannot solely account for the elevated protein
breakdown or skeletal muscle wasting in cancer patients. Total parenteral nutrition and
branch-chained amino acid supplementation (valine, isoleucine and leucine) does not
necessarily increase skeletal muscle protein stores nor does it improve survival times in
cancer patients (Argiles, 1997). In patients with decreased energy intake (due to loss of
appetite or anorexia), a commensurate reduction in total body energy expenditure is not
11
always evident. In fact, Hyltander and colleagues (1991) have shown that cancer patients
have a higher resting energy expenditure compared with normal, healthy controls.
Several potential mechanisms could lead to higher resting energy expenditure in cancer
patients including increased brown adipose tissue activation via nonshivering
thermogenesis (Bianchi, 1989), increased lactate recycling to replenish glucose stores
(Argiles, 1997), or abnormal function of Na+/K+-ATPase pump in tumor cells (Racker,
1976).
Many pro-catabolic chemical signals arise from the tumor proper and from the
deleterious side effects of conventional anti-neoplastic therapy. These two phenomena
exacerbate the production of various inflammatory cytokines including interleukin-1 (IL-
1), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) and release several cancer-
specific cachectic factors (Argiles, 1997; Lorite, 2001; Fearon 2002).
Several substances initiate the degradation of adipose and skeletal muscle (Figure 1).
The best characterized is TNF-α, which is secreted from infiltrating macrophages and
inhibits lipoprotein lipase (LPL). Inhibition of LPL depletes body lipid stores and
releases free fatty acids into the blood stream. These free fatty acids can be oxidized and
used to support tumor cell protein synthesis and growth. TNF-a has also been associated
with nuclear factor-κB (NFκB) activation in C2C12 myocytes (Guttridge, 2000). NFκB
antagonizes myoD protein expression, inhibits skeletal muscle differentiation and
interrupts the development of nascent muscle fibers. Interestingly, rats trained to run on a
treadmill display reduced plasma TNF-α levels in response to lipopolysaccharide
challenges that induce inflammation (Bagby, 1994). This response potentially attenuated
the ability of the macrophage to respond to further challenges. Alternatively, exercise
might acutely modulate the response of stress hormones and catecholamines that
effectively suppress TNF-α levels (Beutler, 1986).
The most recently discovered factor involved in cachexia-induced skeletal muscle
degradation is proteolysis- inducing factor (PIF) (Lorite, 1998). PIF has been isolated
from the urine of cancer patients (Baracos, 2000) and from tumor cells in vitro (Todorov,
12
1997). Purified PIF introduced into C2C12 myoblasts drastically increases skeletal
muscle degradation (~1.5 fold greater than controls) in a dose-related matter with a
maximal effect between 2-4 nM (Lorite, 2001). This effect of elevated skeletal muscle
breakdown is attenuated with addition of monoclonal antibody raised against PIF.
Furthermore, therapeutically blocking PIF with eicosapentaenoic acid (EPA) attenuates
the development of cachexia and allows for the accumulation of lean body mass (Hussey,
1999; Tisdale, 2001).
Macrophages
Liver
Tumor Skeletal Muscle
↑ Protein Degradation
PIF
TNF-α IL-1 IFN-γ
Gluconeogenic Pathways
Glucose
Pyruvate Lactate
Krebs Cycle
Figure 1. Metabolic interactions between tumor and host. There are three main metabolic disturbances associated with cancer cachexia: (1) increased lactate recycling between tumor and liver (Cori Cycle); (2) lipid mobilization (not depicted); and (3) muscle wasting stimulated by tumor-derived PIF or by various cytokines released from infiltrating macrophages. Because of enhanced skeletal muscle protein degradation, gluconeogenic amino acids are released and taken up by the liver to form glucose or taken up by the tumor to sustain both energy and nitrogen demands of the growing mass. TNF-α, Tumor Necrosis Factor-α; IL-1, Interleukin-1; IFN-γ, Interferon-γ; PIF, Proteolysis-Inducing Factor
Alanine
Glutamine
Cori Cycle
13
Cancer Cachexia: Effect on Skeletal Muscle Protein Turnover
Beyond its motile function, skeletal muscle serves as a large protein reservoir that can be
mobilized in stressful states (e.g., cancer cachexia) as a substrate source for energy
metabolism. Skeletal muscle wasting associated with cancer cachexia results in a large
release of amino acids, particularly alanine and glutamine, that can be used as substrates
for gluconeogenesis or to support Krebs Cycle function, respectively (Figure 1).
Lysosomal proteases are thought to be solely responsible for degradation of membrane
proteins and certain soluble proteins in normal muscle (Attaix, 1998). Although purified
lysosomal cathespins B, D, H and L have been shown to degrade purified actin and
myosin in vitro to some degree (Bird, 1980), Lowell and colleagues (1986) have clearly
demonstrated that lysosomes do not play a role in myofibrillar protein turnover in vivo.
The calpain protease system and the ATP-dependent-ubiquitin proteasome system are the
primary pathways that regulate skeletal muscle degradation and myofibrillar protein
turnover (i.e., actin and myosin). Specific skeletal muscle proteins targeted for calpain-
mediated degradation include Z disk proteins, tropomyosin, troponin T, troponin I,
desmin, titin, nebulin and C-protein. Degradation of these structural proteins help to
liberate actin and myosin filaments from the sarcomere. Free actin and myosin are
subject to ATP-dependent, ubiquitin-mediated proteolysis. Thus, the calpains only
initiate myofilament turnover by releasing these proteins from the sarcomere and do not
degrade actin or myosin into their constituent amino acids.
The majority of intracellular proteolysis in skeletal muscle occurs through the ATP-
dependent-ubiquitin-proteasome system. After the calpain protease system dissociates
the myofibril complex, the free actin and myosin molecules are poly-ubiquitinated. Poly-
ubiquitin chains provide a signal that targets the substrate for degradation by the 26S
proteasome complex and releases amino acids into the myoplasm (see Figure 22 in
appendix) (Attaix, 1998).
14
Perhaps not surprisingly, muscles from tumor-bearing rats display increased calpain
activity levels, decreased calpastatin activity levels (an endogenous inhibitor of calpain)
and an increased calpain/calpastatin ratio (Temparis, 1994; Baracos, 1995; Costelli,
2001). Cachectic rats and humans with enhanced skeletal muscle proteolysis display
concomitant increases in levels of ubiquitin-protein conjugates (Llovera, 1994; Lorite,
1998; Schwartz, 1999) and dramatic increases in ubiquitin and proteasome subunit
mRNA levels (Baracos, 1995). In all, increased skeletal muscle protein degradation in
cachectic cancer patients can partially be attributed to increased activity of these two
catabolic systems.
Cancer Cachexia: Effect on Skeletal Muscle Physiology
Because muscle force is directly related to the amount of myofibrillar proteins acting in
parallel within the muscle’s fibers, increased protein turnover as evident in cancer
cachexia could dramatically alter force production. Yet, little is known about the effect
of cachexia-induced decreases to muscle mass on other aspects of muscle function such
as force-velocity characteristics, power output, metabolic properties, MHC isoform
compositions and fatigability.
MHC isoform composition is a common marker of skeletal muscle physiology. MHC is
a major component of the skeletal muscle contractile protein, myosin. Mature human
skeletal muscle might contain up to three different MHC isoforms, types I, IIa and/or IIx,
each associated with categorical shortening velocities, force and power outputs,
metabolic pathways and substrate concentrations for energy production (Table 1).
Mature rat skeletal muscle expresses a MHC isoform with higher myosin ATPase activity
than human MHC type IIx. Thus, rats contain a fourth MHC isoform, termed MHC type
IIb, that is structurally and functionally distinct from MHC type IIx.
In general, MHC isoforms range from slow, oxidative (MHC type I) to fast, glycolytic
(MHC type IIx and IIb) types. Due to higher concentrations of glycolytic enzymes and
higher rates of ATPase activity (i.e., myosin ATPase and sarcoplasmic reticulum
15
ATPase), muscles containing a predominance of fast MHC isoforms generally reach
fatigue before muscles containing predominantly slow MHC isoforms (Kraus, 1994).
Therefore, the distribution of MHC isoform types (i.e., slow vs. fast isoforms) and the
metabolic parameters associated with each type are directly related to muscle fatigability.
Parameter MHC type I MHC type IIa MHC type IIx
Force (mN) Low (0.60) Moderate (0.89) High (1.11)
Shortening Velocity
(fiber lengths/sec.) Slow (0.58) Moderate (2.95) Fast (4.72)
Peak Power (µN*FL-1*s-1)
Low (8.3) Moderate (49.1) High (115.2)
Mitochondrial density
High High Low
Glycogen granules Few Many Many
Endurance Fatigue Resistant
Moderately Fatigable Highly Fatigable
Table 1. Differences between MHC fiber types in human skeletal muscle. Data appearing in the table taken from Widrick, 2002 and are values recorded using skinned muscle fibers. Skeletal muscle is referred to as a “plastic” tissue, meaning that proteins that regulate
contraction and metabolism can alter isoform type protein content depending on the
pattern of electrical activity the muscle receives. For example, reduced neuromuscular
activity due to spinal cord transection (Talmadge, 1995) or spinal cord isolation (Jiang,
1991) or reduced body weight bearing through hindlimb suspension (Bigard, 1997) or
space flight (Ohira, 1992) increases the percentages of fast fiber-like protein phenotypes
(e.g., increased fast MHC isoforms at the expense of slower MHC isoforms). In contrast,
increased neuromuscular activation due to tonic, low frequency stimulation (Pette, 1985)
or FO (Dunn, 1999) induces slow fiber- like protein phenotypes.
Skeletal muscle plasticity might involve a change in the amount of MHC protein, the type
of MHC protein expressed (i.e., isoform content), or a combination of the two. Myosin is
16
a common protein used to examine the molecular and cellular events underlying muscle
plasticity for several reasons. First, myosin is a structural and regulatory protein
comprising about 25% of the total protein pool. It forms the structural backbone of the
contractile apparatus and drives the conversion of chemical energy (ATP) into
mechanical work. Secondly, myosin is expressed as different isoforms, each with
categorical rates of contraction and functional capacity. Myosin is expressed in constant
proportion to other muscle proteins. For instance, during hypertrophy, myosin content
increases as fiber cross-sectional area increases. During atrophy, myosin content
decreases as fiber cross-sectional area decreases. Lastly, the regulation of myosin
expression seems to be highly controlled by factors that have an impact on
transcriptional, translational and post-translational processes (for review see Baldwin,
2002).
Although the amount and pattern of neuromuscular activity contributes to the isoform
type of MHC expressed, neural activity is not the sole regulator of MHC isoform protein
expression. In fact, small subpopulations of fibers in soleus muscle appear not to be
influenced by electrical activity at all. For instance, cat soleus muscle still contains a
population of MHC type I fibers six months after spinal cord isolation, an experimental
model that “silences” all electrical activity from the motor neuron and is associated with
dramatic slow-to-fast MHC isoform transitions (Talmadge, 1996b). Altered expression
patterns of MHC fiber phenotypes have also been linked to mechanisms independent of
altered neuromuscular activity. Genetic factors intrinsic to muscle, neural activity-
independent factors (i.e., neurotrophic factors), mechanical factors, metabolic factors and
hormonal factors have been implicated in altering MHC isoform composition (for review
see Edgerton, 2002). For example, levels of circulating hormones potentially regulate
MHC isoform protein expression. Reductions to circulating growth hormone in
hypophysectomized rats decrease MHC type I and MHC type IIa mRNA levels and
increase MHC type IIb mRNA levels (Laughna, 1994). Also, thyroid hormone status
affects MHC isoform expression. Hypothyroidism is consistently associated with fast-to-
slow MHC isoform transitions (Pette, 2000; Baldwin, 2001). Conversely,
hyperthyroidism increases the percentage of fast MHC fiber types (Caiozzo, 1991). Our
17
laboratory has reported increased MHC type IIx by ~14% in plantaris muscle from CHF
rats compared to controls (Spangenburg, 2002) potentially due to increases in circulating
TNF-α levels (Dalla Libera, 2001).
Interestingly, slow-to-fast MHC isoforms shifts have been reported in soleus muscles of
mice bearing an adenocarcinoma tumor (Diffee, 2002). However, current work in our
laboratory fails to corroborate shifts to MHC isoform expression in the soleus muscle
(Otis, unpublished observations). In fact, we have found no change to MHC isoform
distribution in soleus muscles 2, 4 or 6 weeks after implantation of Morris hepatoma MH-
7777 cells. This discrepancy might be due to the different animal models used (mice vs.
rat), the different cell lines used to induce tumor formation (murine C-26 adenocarcinoma
vs. MH-7777 hepatoma cells) and/or the level of physical activity of the animals.
Because the soleus is the primary load-bearing muscle of the plantar flexor group, the
physical tumor burden may have provided a significant hypertrophic stimulus that
preserved soleus muscle mass and eliminated a potential slow-to-fast MHC isoform
transition. Although Diffee and colleagues (2002) did not analyze diaphragm muscles,
data from our rats indicate that respiratory dysfunctions linked to cancer cachexia are not
due to shifts toward a more fatigable MHC profile, but may be directly attributable to
increased skeletal muscle proteolysis and atrophy (Otis, 2003).
Strength training increases the electrical activity received by the exercising muscle and
causes the contractile (i.e., MHC isoform composition) and biochemical (i.e., metabolic
substrate utilization) properties of characteristically fast muscle to become more like slow
muscles (Baldwin; 1982, Roy, 1982). While it is known that strength training can
attenuate skeletal muscle atrophy in cachectic cancer patients (Kasper, 2000; Evans,
2002), it is unknown if cancer cachexia would interfere with a training- induced transition
toward a slow-fiber like phenotype in the plantaris muscle. Recently, the serine/threonine
protein phosphatase, calcineurin, has been linked to molecular pathways that regulate
skeletal muscle mass and fiber-type specific protein expression (Dunn, 1999; Musaro,
1999; Semsarian, 1999; Olson, 2000; Talmadge, 2000a; Otis, 2001).
18
Calcineurin
Calcineurin, also known as protein phosphatase 2B (PP2B), is a serine/threonine,
Ca2+/calmodulin-dependent protein phosphatase found at concentrations 10-fold higher in
brain and skeletal muscle than in other tissues (Klee, 1978). In addition to calcineurin,
the serine/threonine protein phosphatases include protein phosphatases 1 (PP1), 2A
(PP2A) and 2C (PP2C). This family of phosphatases are essential for a number of signal
transduction pathways in eukaryotic cells (for review see Rusnak, 2000). Type-1 protein
phosphatases dephosphorylate the a-subunit of phosphorylase kinase distinguishing it
from type-2 protein phosphatases, which dephosphorylate the ß-subunit of phosphorylase
kinase. A difference in divalent metal cation dependence distinguishes type-2 protein
phosphatases. PP2A was originally described as having no divalent metal cation
requirement for activation, while PP2C was found to be Mg2+-dependent and PP2B
(calcineurin) to be Ca2+/calmodulin-dependent (Cohen, 1989).
Calcineurin exists as a heterodimer, composed of a 59 kDa catalytic subunit (CaNα) and
a 19 kDa Ca2+-binding regulatory subunit (CaNβ) (Figure 2). CaNα contains an amino-
terminal catalytic domain and several domains near the carboxyl terminus that confer
regulatory function, including the calmodulin-binding domain, an autoinhibitory domain
that binds in the active site cleft in the absence of Ca2+ and the CaNβ-binding domain.
Four Ca2+ ions bind with high affinity [dissociation constant (Kd) ≤ 10-6 M] to Ca2+-
binding domains with “EF-hand” motifs located on the CaNβ subunit (Klee, 1976).
While both subunits are required to interact for functional, Ca2+/calmodulin-dependent
phosphatase activity, deletion of the carboxyl-terminal regulatory region of CaNα
removes the Ca2+ requirement for activation and results in a constitutively active enzyme
(Wu, 2000).
19
During periods of sustained elevations of intracellular Ca2+, calcineurin is activated and
dephosphorylates nuclear factor of activated T cells c1 (NFATc1). The N-terminal ~400
residues of NFATc1 serve as a calcineurin-activated regulatory domain and contains
several phosphorylated serine residues that mask the nuclear location sequence (NLS).
The dephosphorylation of NFATc1 exposes the NLS and the DNA recognition region in
the DNA-binding domain (for review see Rao, 1997). In skeletal muscle, nuclear
NFATc1 binds to conserved DNA sequences known as NFATc1 response elements
(NRE) and activates the transcription of several growth regulatory genes and fiber type-
specific genes (Chin, 1998; Wu, 2000; Olson, 2000). Nuclear NFATc1 can be
rephosphorylated by glycogen synthase kinase-3β (GSK-3β) to signal nuclear export and
terminate calcineurin-mediated gene transcription (Vyas, 2002) (Figure 3).
100 300 400 200 500
N-
Term
Catalytic
CM
CaNß
Binding
AI
Calcineurin-a Domain Structure
Calcineurin-ß Domain Structure
E F
E F
E F
E F
Figure 2. Calcineurin Domain Structures. The catalytic domain of CaNa is located in the amino-terminal followed downstream by three regulatory domains. CaNß contains four Ca2+-binding EF hand motifs. CaN, calcineurin; CM, calmodulin; AI, autoinhibitory; EF, EF hand motifs. Scale represents amino acid number.
20
Calcineurin is the primary target of the immunosuppressant drugs cyclosporin A (CsA)
and tacrolimus (FK506), which are commonly prescribed following organ transplantation
in attempts to minimize tissue rejection (Schafer, 2002). These drugs bind to cytoplasmic
receptor proteins, cyclophilin A and FKBP 12, respectively, at the interface between
CaNα and CaNβ . These drug-receptor complexes sterically block access to the catalytic
site on CaNα and effectively prevent the activation of calcineurin-dependent genes (Liu,
1991; Kawamura, 1995). CsA has been used extensively to demonstrate a potential role
of calcineurin in Ca2+-dependent signal transduction pathways in skeletal muscle
(Musaro, 1999; Dunn, 1999, 2001; Mitchell, 2002). Administration of CsA attenuates
the up-regulation of slow fiber-specific gene promoters in the rat soleus (Chin, 1998) and
blocks the fast-to-slow MHC transition in FO mouse plantaris muscle (Dunn, 1999).
Although these drugs are commonly used to block calcineurin activity, results obtained
from studies that administer CsA are controversial. CsA treatment in mice that have
undergone reloading of the plantaris muscles following hindlimb suspension not only
prevents regrowth, but also reduces plantaris mass below values reported in vehicle-
treated animals. In addition, muscle mitochondrial respiration is decreased due to
suppression of mitochondrial electron chain capacity following 2 weeks of CsA
administration (Hokanson, 1995; Mercier, 1995). Because skeletal muscle oxidative
capacity increases in coordination with slow MHC isoforms following endurance training
(Margaritis, 1997), altered mitochondrial respiration secondary to CsA administration
might influence MHC isoform expression. Prolonged CsA treatment has also been
implicated in altering the cardiovascular system, including cardiotoxicity (Bellamy,
1995), altered characteristics of Ca2+-release channel in cardiac sarcoplasmic reticulum
(Park, 1999), reduced capillary density (Breill, 1999), reduced IGF-1 signaling (Grounds,
2002) and reduced systolic function (Kingma 1991). Our lab has shown that reductions
to cardiac function following myocardial infarction can alter skeletal muscle MHC
composition (Spangenburg, 2002). Thus, changes to muscle gene expression attributed to
calcineurin activity may be a secondary result of CsA side effects on various mechanisms
intrinsic and extrinsic to skeletal muscle.
21
Skeletal Muscle Contraction and Calcineurin Signaling
Following motor nerve depolarization, acetylcholine (Ach) is released from the motor
neuron terminal into the synaptic cleft located between the muscle and nerve. Ach binds
to Ach receptors integrated into the muscle membrane in a region known as the motor
end plate. Ach binding results in the depolarization of the muscle fiber sarcolemma. An
action potential propagates along the sarcolemma via sequential depolarization of
neighboring, voltage-gated Na+/K+ channels (Ebashi, 1976). The action potential enters
invaginations of the muscle membrane known as t-tubules and interacts with the
NFATc1
Contractile
Activity
Calcium
CaN
NFATc1
NFATc1-P
NFATc1
NFATc1-P
CaN? GSK-3β
(+)
(+)
(-)
GATACACC TACGGATC CTATGTGG ATGCCTAG NRE
NUCLEUS MYOPLASM
Figure 3. Calcineurin-mediated signaling pathway. Once dephosphorylated by CaN, nuclear factor of activated T cells c1 (NFATc1) translocates into the nucleus along with CaN where they can bind to a conserved DNA binding sequence known as the NFAT response element (NRE). Once bound to the NRE, NFATc1 appears to induce transcription of slow-muscle specific genes and growth regulatory genes. Nuclear NFATc1 can be rephosphorylated by glycogen synthase kinase-3β (GSK-3β) signaling nuclear export and reducing CaN-mediated gene activation. CsA, Cyclosporin A; FK-506, Tacrolimus; NFATc1-P, phosphorylated form of NFATc1.
CsA/FK-506
(-)
22
sarcolemma-bound, voltage-gated dihydropyridine receptor (DHPR). DHPR provides a
physical link to the calcium release channel (ryanodine receptor; RyR) on the
sarcoplasmic reticulum (SR). Calcium released through the RyR binds to the thin
filament-associated protein troponin C. This exposes myosin-binding sites on actin and
facilitates crossbridge interaction and subsequent force generation (Figure 4).
In addition, the amount of Ca2+ released from the SR into the myoplasm depends on the
amount and pattern of electrical activity a muscle receives. This pattern of electrical
activity is received in a fiber type-specific pattern. Slow motor neuron- like activity (~
sustained 10 Hz stimulation) results in myoplasmic Ca2+ concentrations ([Ca2+]i) between
100 and 300 nM (Chin, 1996), whereas fast motor neuron- like activity results in
intermittent spikes of Ca2+ approaching 1 µM (Westerblad, 1991).
The initial binding of Ca2+ to troponin C accounts for the sigmoidal relationship between
[Ca2+]i and force generation (for review see Williams, 1995). During muscle relaxation,
myoplasmic calcium is resequestered into the SR via the SR-Ca2+-ATPase pump
(SERCA). Therefore, [Ca2+]i fluctuates with the state of muscle contraction or
relaxation. The fluctuations of Ca2+ during muscle contraction/relaxation cycles are
intriguing as potential initiators of the Ca2+/calmoduling-dependent calcineurin signaling
pathway.
In mature skeletal muscle, calcineurin is tethered to the Z-disc structures of sarcomeres
through its association with a novel structural protein called calsarcin (Frey, 2000; Liu,
2001). Because calcineurin is localized to Z-disc structures, it is in close proximity to the
Ca2+-release channel (RyR). This microenvironment may provide a potential link
between calcineurin activation and regulated Ca2+ release. Moreover, this localization of
calcineurin to SR luminal Ca2+ reserves potentially couples muscle contractile activity to
transcriptional activation of fiber type-specific and growth-dependent genes.
23
TTrrooppoommyyooss iinn
NNeeuurroo mmuuss ccuu llaarr JJuunncctt iioonn
Ca2+ Ca2+
AAcctt iioonn PPootteenn tt iiaall
SSaarrccoo llee mmmmaa
TTrraannss vveerrss ee TTuubbuullee
CCaa22++-- AATTPPaass ee
TTrrooppoonniinn Complex
AAcctt iinn
MMyyooss iinn
AAccttoommyyooss iinn AATTPPaass ee
CCaa22++ CCaa22++
CCaa22++
AAcchh
RRyyRR
DDHHPP RR
Figure 4. Overview of Muscle Contraction. Depolarization of the motor neuron releases Ach into the neuromuscular junction where it binds to Ach receptors on the sarcolemma membrane. These receptors initiate an action potential that travels along the sarcolemma and propagates into t-tubule invaginations. The action potential stimulates physical interaction between DHPR-RyR releasing Ca2+ from the SR into the myoplasm. Myoplasmic Ca2+ binds to troponin C turning the troponin complex that allows actin and myosin to interact resulting in ATP-dependent crossbridge cycling. During cessation of action potential firing (i.e., relaxation) Ca2+ is shuttled back into the SR via the Ca2+-ATPase pump. Ach, Acetylcholine; RyR, Ryanodine receptor; DHPR, Dihydropyridine receptor.
24
Calcineurin: Role in Skeletal Muscle Protein Expression and Hypertrophy
Skeletal muscle increases in size when repeatedly forced to support masses greater than
normally experienced (e.g., strength training, FO). However, the molecular mechanisms
that link this mechanical signal to the chemical signals that regulate gene expression
remain largely unknown. A growing body of evidence suggests that calcineurin-
mediated pathways regulate skeletal muscle hypertrophy and fiber type-specific protein
expression through the dephosphorylation of transcription factors, including NFATc1 and
myocyte enhancer factor-2 (MEF2) (Dunn, 1999; Musaro, 1999; Semsarian, 1999; Olson,
2000; Talmadge, 2000b).
We have shown that plantaris fibers from transgenic mice expressing a constitutively
active form of calcineurin (MCK-CN*) have an increased percentage of fibers staining
positively for slow isoforms of MHC, troponin I (TnI) and sarcoplasmic reticulum Ca2+-
ATPase (SERCA) (Otis, 2001). Interestingly, the isoform expressions of these proteins
were coordinated, such that a fiber would contain either all slow or all fast isoforms
(Figure 5). Therefore, calcineurin might influence the transcription of seve ral fiber-type
specific genes. Plantaris fibers from these MCK-CN* mice also display increased
oxidative capacity and reduced glycolytic capacity, characteristics common to slow fibers
(Otis, 2002).
25
Figure 5. Immunohistochemical staining of MCK-CN* plantaris fibers. Serial sections from plantaris muscle were cut at 10µm and incubated in primary antibodies specific for slow or fast isoforms of MHC, TnI and SERCA. Note that fiber #1 stains only for the slow isoforms of MHC, TnI and SERCA. In contrast, fiber #2 stains only for the fast isoforms of these proteins. Primary antibodies: A, MHC type I; B, MHC types IIa, IIx, IIb (fast); C, SERCA type 2 (slow); D, SERCA type 1 (fast); E, TnI slow; F, TnI fast
26
Recently, the activation of MEF2 transcription factors has been associated with
calcineurin-dependent signaling pathways (for review see Black, 1998). Calcineurin and
MEF2 form a physical complex in the nucleus that aids to hypophosphorylate MEF2.
This complex augments the potency of the transcriptional activation domain of MEF2. In
turn, MEF2 activation has been shown to increase myoglobin content and slow troponin I
gene expression in skeletal muscle (Wu, 2001). Furthermore, nuclear NFAT has been
associated with the activation of several transcription factors, including MEF2, AP-1 and
GATA2 (Black, 1998; Musaro, 1999), which are known to be involved in hypertrophic
signaling cascades (for review see Olson, 2000).
Insulin- like growth factor (IGF-1), a potent hypertrophic mitogen, appears to work
through calcineurin-dependent pathways in vitro in association with GATA2
transcription factors (Musaro, 1999). Post-mitotic skeletal myocytes transfected with
IGF-1 co-localize GATA2 and NFATc proteins in a nuclear subset of hypertrophic
myocytes. In addition, calcineurin and GATA2 mRNA transcripts are up-regulated and
calcineurin protein accumulates within the nucleus. However, Rommel and colleagues
(2001) suggest that calcineurin does not mediate IGF-1-induced hypertrophy, but
unexpectedly acts via Akt/mammalian target of rapamycin (Akt/mTOR) pathways to
antagonize calcineurin signaling during myotube hypertrophy.
The muscle regulatory factors (MRF) are a large family of transcription factors that
include MyoD, Myf5, myogenin and MRF4. MRFs contain a basic region that mediates
DNA binding to an E-box consensus DNA element and a basic helix- loop-helix (bHLH)
domain that mediates dimerization (Davis, 1990). MRF activation has been shown to
regulate skeletal muscle differentiation and myotube formation (for review see Megeney,
1995). Recent evidence suggests that MRF proteins regulate growth of existing
myofibers (Lowe, 1999) and fiber-type specific protein expression (Hughes, 1999).
Specifically, Myf5 mRNA levels are increased in mononucleated reserve cells in
myotube cultures treated with the calcium ionophore ionomycin (Friday, 2000). The
increased transcription of Myf5 is dependent on the activity of calcineurin and NFAT,
and can be specifically enhanced by overexpressing the NFATc isoform.
27
Inhibition of GSK-3β in vitro stimulates myotube hypertrophy presumably by increasing
the nuclear/myoplasmic NFATc1 concentration ratio (Vyas, 2002). Furthermore,
resistance exercise decreases GSK-3β activity suggesting a greater nuclear NFAT1c
accumulation and sustained opportunities for growth and fiber type-specific gene
regulation (Markuns, 1999). GSK-3β has also been identified as an antagonist of cardiac
hypertrophy (Haq, 2000; Antos, 2002), which suggests a potential calcineurin-dependent
pathway mediating cardiac hypertrophy (Molkentin, 1998; Sussman, 1998). Nuclear
NFATc1 protein accumulation appears to be a key step in the regulation of skeletal
muscle and cardiac muscle growth. However, other reports fail to corroborate
calcineurin/NFATc1-dependent pathways as leading factors regulating muscle growth or
fiber type-specific gene expression (Calvo, 1999; Swoap, 2000; Rommel, 2001).
Swoap and colleagues (2000) cotransfected C2C12 myotubes with constitutively active
calcineurin and one of several different muscle-specific or non-muscle specific reporter
plasmids. Interestingly, overexpression of activated calcineurin induced expression of
the fast-type muscle-specific isoforms of phosphoglycerate mutase (PGAM-M), SERCA1
and MHC IIb promoters, the slow-type myosin light chain 2 (MLC2) and MHC I
promoters, and the non-fiber type-specific skeletal a-actinin and NFATx3 promoters.
Therefore, activated calcineurin in cell cultures might induce muscle-specific promoters
in a non-fiber type-specific fashion. In addition, CsA treatment in vivo did not induce
changes in MHC proportions (Biring, 1998) nor prevented hypertrophy in transgenic
mice overexpressing IGF-1 (Musaro, 2001) or in exercised rats following spinal cord
transection (Dupont-Versteegden, 2002). Obviously, a potential role of calcineurin
mediating growth and fiber-type specific gene expression warrants further exploration.
28
Role of Myonuclear Apoptosis in Skeletal Muscle Atrophy
Skeletal muscle fibers are multinucleated cells that may contain hundreds to thousands of
myonuclei within a common cytoplasm. Multiple nuclei supplying a single fiber help to
improve intracellular diffusion and reduce translation limitations of nuclear products.
Therefore, each myonucleus within a fiber is responsible for maintenance of a specific
and limited cytoplasmic region. This region is referred to as the “DNA unit” (Cheek,
1985) or “myonuclear domain” (Hall, 1989).
Myonuclear number is reduced in a variety of skeletal muscle atrophy models including
hindlimb suspension (Allen, 1997), spaceflight (Allen, 1996) and spinal cord injury
(Allen, 1995) and might partially explain the decrease in fiber size. During atrophy,
when the demands for mRNA transcription and protein trans lation are attenuated (Babij,
1988), myofibers respond by eliminating myonuclei. The elimination of myonuclei
during atrophy might allow for maintenance of a constant myonuclear domain.
The mechanisms by which these nuclei disappear are not clearly understood, but
programmed cell death, or apoptosis, might play a significant role. Cellular apoptosis is
characterized by a series of morphological changes triggered by events outside the cell or
within the mitochondria, leading to the activation of caspases and subsequent cell death.
These changes include targeted chromatin compaction, cellular condensation, budding to
produce enveloped apoptotic bodies and cellular destruction without inflammation
(Wyllie, 1980).
However, skeletal muscle tissue is unique because its myofibers are multi-nucleated cells
and can undergo individual myonuclear apoptosis without complete cell death (for
review, see Primeau, 2002). Therefore, myonuclear apoptosis represents a “modified”
version of classic apoptosis as programmed nuclear death is achieved without
unavoidable cell death. Interestingly, heterokaryons formed by the fusion of several uni-
nucleated cells can house a subpopulation of apoptotic nuclei. These apoptotic nuclei do
not affect the survival of other nuclei within the common cytoplasm or destroy the cell
itself (Dipasquale, 1992). However, it is unclear if the mechanisms involved in apoptosis
29
can account for the selected degradation of myonuclei. In fact, increased myonuclear
apoptosis in dystrophic mdx mice has been linked to perforin-mediated myonuclear
killing by cytotoxic T lymphocytes (Spencer, 1997).
Role of Myonuclear Accretion in Skeletal Muscle Hypertrophy
In general, skeletal muscle hypertrophy is the result of a positive nitrogen balance and net
protein deposition (i.e., protein synthesis exceeds protein degradation). Increased protein
synthesis can be accomplished by increasing the myonuclear domain of each myonucleus
without changing the total number of myonuclei. This would require each myonucleus to
encode more proteins to supply a larger cytoplasmic volume. Alternatively, the increased
deposition of proteins could be achieved by maintaining the level of RNA synthesis per
myonuclei at a normal level and increasing the total number of myonuclei. This strategy
would maintain or decrease the size of this myonuclear domain for each myonucleus.
The majority of research indicates that skeletal muscle fiber hypertrophy is accomplished
by increasing total myonuclei number (through myonuclear accretion) such that
myonuclear domain size remains constant (Allen, 1995; McCall, 1998).
Satellite cells, also known as muscle precursor cells or muscle stem cells, provide the
largest pool of new myonuclei for hypertrophying fibers (Moss, 1971; Schiaffino, 1976;
Darr, 1989). Quiescent satellite cells lie between the sarcolemma of a muscle fiber and
the basal lamina and can enter the cell cycle and fuse with the fiber proper (Joubert,
1989). Inhibition of satellite cell proliferation via gamma irradiation prevents the satellite
cell fusion and myonuclear accretion necessary for skeletal muscle fiber hypertrophy
and/or repair (Rosenblatt, 1992; Barton-Davis, 1999; Mitchell, 2001).
Recently, it has been suggested that calcineurin modulates the response of satellite cells
to hypertrophic stimuli in a muscle-type dependent fashion (Mitchell, 2002). The slow
soleus muscle regrows in a bimodal fashion when subjected to the hindlimb
suspension/reloading model, a common technique used to induce skeletal muscle atrophy
(hindlimb suspension) followed by hypertrophy (reloading). The initial phase of this
30
bimodal response appears to be independent of calcineurin signaling pathways and occurs
without myonuclear accretion. The second phase of regrowth involves myonuclear
accretion and appears to rely on calcineurin pathways. Conversely, fast plantaris muscle
appears to be entirely regulated by calcineurin-dependent processes following hindlimb
suspension and reloading (Figure 6).
ATROPHY
CsA
(-) (-)
GROWTH
CsA
(-)
A
B
MPC
Figure 6. CaN-dependent and CaN-independent stages during regrowth. A, Soleus muscle loses myonuclei following hindlimb suspension. Regrowth occurs in two stages: (1) a CaN-independent stage followed by, (2) CaN-dependent stage that requires the addition of new myonuclei. In this latter stage, CsA administration blocks regrowth B, Plantaris muscle does not lose myonuclei following hindlimb suspension, but subsequent regrowth following reloading appears to be mediated by CaN-dependent pathways. CaN, calcineurin; MPC, muscle precursor cells; CsA, cyclosporin A.
(1) (2)
31
Summary
Cancer cachexia is a debilitating syndrome that produces severe skeletal muscle wasting.
The chemical signals for increased skeletal muscle proteolysis arise from the tumor
proper and infiltrating macrophages and often leave the cachectic patient with asthenia,
generalized weakness, an inability to tolerate chemotherapy treatments and decreased
survival times. Although strength training, diet supplementation and growth hormone
treatments attenuate the degree of muscle atrophy, these interventions are often difficult
to prescribe considering constraints of conventional chemotherapy such as asthenia and
nausea. The degree of skeletal muscle atrophy could be attenuated the quality of life
might be enhanced for patients in late-stage malignancy.
Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase potentially plays a pivotal
role in downstream regulation of skeletal muscle mass and fiber type-specific gene
expression. Exploitation of the calcineurin signaling pathway through gene therapy
approaches may minimize the degree of muscle atrophy, increase the percentage of slow
MHC fiber types that mimic trained muscle and ultimately improve survival times of
cachectic cancer patients.
32
CHAPTER THREE - METHODS
33
Study I: Morphological changes to skeletal muscles from tumor-bearing rats
The current project was completed in two separate studies (termed Study I and Study II).
Study I tracked morphological changes to skeletal muscles from tumor-bearing rats after
2, 4 or 6 weeks of tumor development compared to healthy controls. In Study I, body
mass, tumor mass and ATP-independent proteasome activities were assessed to identify
the incubation time necessary for MH-7777 cells to produce the greatest changes to body
and skeletal muscle masses.
Study II: Effects of hypertrophic stimulus on cancer cachexia
Study II attempted to attenuate the effects of cancer cachexia by introducing a significant
hypertrophic stimulus (FO) applied concurrently with tumor development. In Study II,
body mass, skeletal muscle mass, MHC isoform composition, myonuclei/fiber ratios,
CaNα and CaNβ subunit protein content and calcineurin phosphatase activity were
assessed in FO and sham-operated plantaris muscles from healthy and tumor-bearing rats.
Animals
The Virginia Tech Animal Care Committee approved all experimental procedures.
Female Buffalo rats (~150-175 g) were purchased from Harlan Sprague Dawley
(Indianapolis, IN). All rats were housed in pairs under a 12:12 light-dark cycle at room
temperature (72 ± 2°C), and food (Harland Teklad 2018 rodent chow) and water were
provided ad libitum. Rats were randomly assigned to one of four groups (n=6/group): (1)
Veh-S (Vehicle- injected, Sham-operated), (2) Veh-FO (Vehicle- injected, FO) (3) TB-S
(Tumor-bearing, Sham-operated) and (4) TB-FO (Tumor-bearing, FO). Body mass (g)
for each animal was obtained once every three days beginning on the first day of
intervention (i.e., surgery) until termination. On days when body masses were
determined, food consumption was monitored by providing a premeasured amount of
food and weighing the remaining food, including any visible food scattered in the cage.
34
Tumor Model
The Morris hepatoma MH-7777 cell line, generously provided by Dr. Bill Huckle
(Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA), was
used to induce tumors. To the author’s knowledge, MH-7777 cell line has not been RAP
tested. Cells for implantation were maintained at 37°C and 5% CO2 in Dulbecco’s
Modified Eagle’s Medium (4.5 g glucose/L, L-glutamine, NaHCO3, and pyridoxine HCl)
supplemented with 10% fetal bovine serum and 50 µg/ml gentamycin. Tumor cells were
trypsinized in a subconfluent state (~85-90% confluency), washed and resuspended in
sterile Dulbecco’s phosphate-buffered saline (PBS) prior to injection. Rats in the tumor-
bearing groups received a suspension of cells (106 cells/300 µl) implanted subcutaneously
on the dorsal side above the hip using a 22-G 1.5” needle. Pilot data indicated that this
cell density and injection location produced solid tumor masses of 6.36 ± 0.93 g after 4
weeks, significantly reduced skeletal muscle masses after 6 weeks and did not alter food
intake (see Figure 21 in appendix). Control rats not receiving MH-7777 cells were
injected with 300 µl of sterile Dulbecco’s PBS solution. Body masses, muscle masses
and tumor masses were obtained at termination.
Ablation Surgeries
The ablation surgeries were performed 5 days prior to the introduction of MH-7777 cells
to avoid the inflammatory phase associated with this surgery (Goldspink, 1983; Timson,
1990) and to help ensure tumor development. A unilateral ablation surgery was
performed so the contralateral leg of each animal could serve as an internal control (i.e.,
FO and non-FO plantaris muscles). Previous work in our laboratory indicated the non-
FO leg in the unilateral FO model could serve as a true control. The animal did not
appear to favor the non-FO leg and the non-FO plantaris muscle did not demonstrate
significant increases in mass compared to those of sham operated controls (Spangenburg,
2002).
35
Animals were anesthetized with a gas mixture of 2 L/min isofluorane and 3 L/min
oxygen. Under aseptic conditions, a midline incision was made through the skin on the
posterior aspect of the animal’s lower leg on the same side as tumor placement. The
distal tendon of the gastrocnemius muscle was isolated from the tendons of its major
synergists (soleus and plantaris) and transected. The distal portion of the gastrocnemius
was removed with care taken not to interrupt the vasculature or innervation leading to the
soleus or plantaris muscles. The skin was closed with 3-0 Ethilon suture. The incision
was treated with a topical antibiotic (Bacitracin). The animals were returned to their
cages and monitored daily for sudden changes in health or temperament (e.g., loss of
appetite, failure to groom) until termination.
Muscle Preparation
Animals were anesthetized with intraperitoneal injections of Ketamine (80 mg/kg) and
Xylazine (10 mg/kg). Muscles were removed, trimmed free of connective tissue and fat,
blotted dry, weighed, frozen in melting isopentane cooled in liquid nitrogen and stored at
-80°C until further analysis. Animals were killed prior to recovery from anesthesia by
removal of the diaphragm muscle.
ATP-independent proteasome activity assay
The ATP-independent proteasome activity assay measures fluorescence following
chymotrypsin- like degradation of the fluorometric substrate, Suc-Leu-Leu-Val-Tyr-AMC
(Bachem). Chymotrypsin cleaves after tyrosine residues and increases fluorescence of 7-
amino, 4-methyl coumarin (AMC). Briefly, fresh soleus, plantaris and diaphragm
muscles were homogenized in ten volumes of buffer containing: 1 M Tris (pH 7.6), 200
mM EDTA and 0.1% β-mercaptoethanol. Samples were centrifuged at 12,000 rpm for
20 minutes at 4°C. Supernatant was subjected to dialysis overnight at 4°C using
Spectra/Por biotech membranes with a retention rating of 15,000 (Spectrum
Laboratories, Inc.). The dialysis buffer contained: 20 mM Tris (pH 7.6), 20 mM NaCl, 1
36
mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT and 20% glycerol. Dialysate was then
centrifuged at 15,200g for 20 minutes at 4°C. A Perkin-Elmer luminescence
spectrophotometer LS-50-B was set to an excitation wavelength of 380-nm and an
emission wavelength of 460-nm. The reaction buffer containing: 1M Tris (pH 8.0),
50µM Suc-Leu-Leu-Val-Tyr-AMC and 0.4% β-mercaptoethanol was warmed to 37°C.
Five-µl dialysate supernatant was added to 1 ml of reaction buffer in a glass cuvette. The
reaction proceeded for 3 minutes and maximal ATP-independent proteasome activity
(AU/min) was measured on the steepest part of the slope over a 20 second duration.
Specific activities were adjusted for total protein content in each sample (AU/min/µg
protein).
Western Blotting for CaNα and CaNβ
Frozen muscles were minced and homogenized on ice in 10 volumes of buffer
containing: 250 mM sucrose, 5 mM EDTA, 100 mM KCl and 20 mM Tris (pH 6.8).
Total muscle protein concentration was determined according to the Bradford method
(Bradford, 1976).
The samples for western blot analysis of CaNα and CaNß subunits were boiled for 2
minutes in sample buffer containing: 480 mM β-mercaptoethanol, 8.75 mM SDS, 7 µM
bromophenol blue, 200 mM Tris-HCl (pH 6.8) and 40% glycerol to a final total protein
concentration of 2 mg/ml. Protein was separated by SDS-PAGE on 10% and 15% total
acrylamide gels for CaNα (~59 kD) and CaNß (~19 kD), respectively, as previously
described (Laemmli, 1970). Forty-µg of total protein were loaded per lane. The proteins
were transferred onto nitrocellulose membranes via the Bio-Rad transblot semi-dry
transfer system. The transfer buffer contained: 1.30 mM SDS, 25 mM Tris-HCl, 192 mM
glycine and 20% methanol. The membranes were washed in Tris-buffered saline (TBS,
pH 7.5) and incubated with monoclonal antibodies raised against the CaNα subunit
(Sigma, 1:10,000 in TBS) or CaNß subunit (Sigma, 1:3,000 in TBS). Primary antibodies
were diluted in 5% nonfat dry milk diluted in TBS overnight at 4°C. An anti-mouse,
37
alkaline phosphatase-conjugated IgG secondary antibody (Sigma, 1:30,000 in TBS) was
applied for 1 hour at room temperature. The membranes were incubated in 5-bromo-4-
chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (BCIP-NBT) to visualize
alkaline phosphatase activity, and then subsequently scanned with an Alpha Innotech IS-
2000 video system. The images were then analyzed with Scion Image software (Scion
Corporation). On each blot, a single plantaris sample from the TB-S group allowed for
semi-quantitative analysis of the CaN subunit signals. This method has been used
previously to control for potential differences in transfer efficiency between membranes
(Spangenburg, 2001).
Calcineurin Phosphatase Activity Assay
Fresh plantaris muscles were homogenized in 0.5 ml of lysis buffer containing: 50 mM
Tris (pH 7.5), 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.2% NP-40. CaN
activity measurements in muscle homogenates were performed according to instructions
provided by Biomol Green Cellular Calcineurin Assay Kit Plus (Biomol Research
Laboratories, Plymouth Meeting, PA). Briefly, muscle homogenates were centrifuged at
150,000 g and desalted using a P6 DG resin to eliminate free phosphate and other
nucleotides (molecular mass exclusion limit of 6 kD). Five-µl aliquots of desalted
effluent were added to buffer containing RII phosphopeptide as a substrate for CaN in
duplicate. Samples were incubated at 30°C for 10 minutes and reactions terminated with
100 µl of Biomol Green reagent. The samples were incubated at room temperature for 30
minutes to allow for color development. The optical density was read at 620 nm (OD620)
on a µQUANT microplate reader. CaN-specific phosphatase activity was determined by
subtracting the OD620 values from the samples with total phosphatase activity from that of
samples incubated in the presence of the calcium chelator, EGTA. The amount of free
phosphate was determined by using a phosphate standard curve run in parallel with the
samples. Data are expressed as nmol PO4 released/ug protein and are normalized to
CaNα protein content.
38
Electrophoretic Separation of MHC isoforms
Frozen muscles were minced and homogenized on ice in 10 volumes of buffer
containing: 250 mM sucrose, 5 mM EDTA, 100 mM KCl and 20 mM Tris (pH 6.8). The
samples were boiled for 2 minutes in buffer containing: 240 mM β-mercaptoethanol, 4.38
mM SDS, 3 µM bromophenol blue, 100 mM Tris-HCl (pH 6.8) and 20% glycerol to a
final total protein concentration of 0.125 mg/ml. MHC isoforms were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously
described (Talmadge, 1993). Briefly, the stacking gel was composed of 30% glycerol,
4% acrylamide-N,N’-methylene-bis-acrylamide (bis) (50:1), 70 mM Tris (pH 6.7), 4 mM
EDTA and 0.4% SDS. The separating gel was composed of 30% glycerol, 8%
acrylamide-bis (50:1), 200 mM Tris (pH 8.8) 100 mM glycine and 4% SDS.
Polymerization of the stacking and separating gels was initiated by adding 0.05%
N,N,N’,N’-tetramethylethylenediamine (TEMED) and 0.1% ammonium persulfate. One-
µg of total protein was loaded per lane and separated in a Biorad mini- transblot at a
constant 70 volts supplied by a Biorad PowerPac 300 for 24 hours. To minimize
temperature fluctuations in the transblot cell, it was kept of ice. The gels were stained
with Coomasie Blue, destained with a solution containing 40% methanol and 10% acetic
acid and analyzed with an Alpha Innotech IS-2000 video densitometric system. MHC
isoform composition was expressed as percentages of the total MHC isoform pool.
Determination of Myonuclear Density
Sample serial sections (8 µm) from the plantaris mid-belly were cut, air dried on gelatin-
coated slides and rehydrated in PBS for 10 minutes at room temperature.
Immunohistochemical detection of dystrophin was used to identify the muscle
sarcolemma. Briefly, dystrophin antibody (Novo Castra, 1:20 in PBS) was applied to the
sections for one hour at room temperature and then washed with PBS. An anti-mouse,
alkaline phosphatase-conjugated IgG secondary antibody (Sigma; 1:50 in PBS) was
applied for one hour at room temperature. Then, the sections were incubated in Fast Red
TR/Naphthol AS-MX solution (Sigma) for detection of alkaline phosphatase activity.
39
To count myofiber nuclei, a Hoechst dye was applied after dystrophin staining. Sections
were fixed in 2% paraformaldehyde and Hoechst-33258 nuclear dye (Sigma) was applied
at 1.2 ng/ml for 30 minutes at room temperature. Sections were viewed with a
fluorescent microscope through an ultraviolet filter (450-nm) and digitized. Between
100-150 fibers per muscle section were analyzed. Cross-sectional areas were not taken
from these muscle sections as the areas are altered due to the experimental procedures
(Dupont-Versteegden, 1999).
Statistics
All data are presented as means ± standard error. A 2-way ANOVA with factor one
being tumor induction and factor two being FO was employed to test for overall
differences followed by Student-Neuman-Keuls post-hoc tests. Pearson’s product
moment correlations were used to correlate tumor mass to the percentage of body mass
lost over six weeks. Potential outliers were determined with the Grubb’s test (Barnett,
1994). Significance was accepted at p ≤ 0.05. Analysis was performed using the
SigmaStat statistical package version 2.03.
Limitations
Studies were limited to us ing only the rat model and Morris hepatoma MH-7777 cells to
induce tumor formation. These two limitations might produce very specific results that
differ from other models and/or cancer producing cell lines. Certainly, the study by
Diffee and colleagues (2002) that used the mouse model and murine C-26
adenocarcinoma cells to induce tumor formation suggests adaptations to MHC isoforms
not evident in the current study. Furthermore, the current study was limited to effects
that occurred within 6 weeks of surgical intervention and/or tumor development.
FO was used to produce adaptations similar to those seen with strength training.
However, by ablating the gastrocnemius, the plantaris chronically supports body weight,
40
whereas, strength training is generally an acute increase in both neuromuscular activity
and load bearing followed by intermittent periods of rest. Additionally, hypertrophy was
assessed by wet mass, which may be influenced by tissue edema and not reflect increased
protein content.
The majority of studies that attempt to link calcineurin-dependent mechanisms to skeletal
muscle hypertrophy or fiber type-specific gene expression use the immunosuppressant
drug CsA to inhibit calcineurin activity (Chin, 1998; Biring, 1998; Dunn, 1999; Bodine,
2001; Mitchell, 2002). However, a recent report suggests that CsA administration
induces phenotypic changes to cancer cells, including invasiveness of non-transformed
cells, by a cell-autonomous mechanism (Hojo, 1999). This report further suggests that
CsA can promote cancer progression by a direct cellular effect that is independent of its
effect on immune cells. Because the Morris hepatoma MH-7777 cell density used for the
current study produces significant tumor masses within 4 weeks and increases the
likelihood of death after 6 weeks (unpublished observations), treating animals with CsA
to block calcineurin activity could potentially shorten life expectancies before the
established experimental time periods elapse. Therefore, calcineurin activity was not
blocked in the current study.
Basic Assumptions
It was assumed that the animals began this study disease and pathogen free and that the
tumor(s) developed were caused directly by the Morris hepatoma MH-7777 cells. It was
also assumed that underlying factors within the muscle or tumor and unknown to the
investigator would not alter any parameter measured (i.e., muscle mass, MHC isoform
composition, calcineurin subunit expression, calcineurin phosphatase activity or
myonuclei/fiber ratios). Furthermore, any changes to these parameters were perceived as
direct consequences of tumor burden and/or FO interventions.
41
CHAPTER FOUR - RESULTS
42
Study I: Morphological Changes to skeletal muscles from tumor-bearing rats
The current project was completed in two separate studies (termed Study I and Study II).
Study I tracked morphological changes to skeletal muscles from tumor-bearing rats after
2, 4 or 6 weeks of tumor development compared to healthy controls. In study I, body and
tumor masses (Table 2 and Figure 8), skeletal muscle masses (Figures 9 and 10) and
ATP-independent proteasome activities (Table 3) were assessed. From study I, it was
determined that 6 weeks of incubation time for Morris hepatoma MH-7777 cells
produced the greatest changes to body and skeletal muscle masses.
Study I: Tumor Masses
The Morris hepatoma MH-7777 cell density used for these experiments (106 cells diluted
in 300 µl of Dulbecco’s PBS) and implanted subcutaneously on the rat’s dorsal side
above the hip produced palpable tumors after ~16-18 days (unpublished observations).
After four weeks of tumor development, final tumor masses ranged from 4.6 to 8.6 g
corresponding to 3.2 and 5.6% of total body mass, respectively. After six weeks of tumor
development, final tumor masses ranged from 0.68 to 24.67 g corresponding to 0.38 and
13.93% of total body mass, respectively (Table 5 in appendix). Tumor masses were
moderately correlated to the percentage change to body mass (r = -0.81; r2 = 0.66)
(Figure 7).
Study I: Body Masses
The major hallmark of cancer cachexia is extreme body mass loss. Body masses when
corrected for tumor masses (henceforth referred to as “adjusted body masses” for tumor-
bearing animals) were significantly reduced after 4 and 6 weeks of tumor growth (Table
2). This loss of mass was not due to changes in eating habits or loss of appetite,
characteristics of anorexia (Figure 8). Therefore, the Morris hepatoma MH-7777 cell line
successfully induced cachectic-like reduction in adjusted body mass after 4 weeks of
tumor development.
43
Study I: Skeletal Muscle Masses
Although adjusted body masses were reduced in tumor-bearing animals after 4 weeks,
skeletal muscle masses were not significantly reduced compared to controls (p ≤ 0.05).
However, there was a strong trend for skeletal muscles from tumor-bearing rats to be
atrophied after 4 weeks. Absolute wet masses of gastrocnemius, tibialis anterior,
extensor digitorum longus, diaphragm and plantaris muscles were reduced after 6 weeks
of tumor growth compared to cont rols (p ≤ 0.05) (Figures 9 and 10). However, relative
muscle masses normalized to adjusted body mass in tumor-bearing rats were similar to
controls, a characteristic of total body wasting.
The soleus muscle from tumor-bearing rats did not atrophy. Because the soleus is the
primary load-bearing muscle of the plantar flexor group, the physical tumor burden
potentially preserved soleus muscle mass by providing a significant hypertrophic
stimulus.
The main proteolytic mechanism involved in skeletal muscle wasting secondary to cancer
cachexia is the ATP-dependent-ubiquitin-proteasome system. Maximal, specific
proteasome activity was elevated in diaphragm and plantaris muscles after 4 and 6 weeks
(p ≤ 0.05), and therefore likely accounted for the reduced diaphragm and plantaris muscle
masses (Table 3). Not surprisingly, proteasome activity in soleus muscles from tumor-
bearing rats was comparable to healthy controls.
44
Study II: Effects of hypertrophic stimulus on cancer cachexia
Data from study I indicate 6 weeks of tumor growth induced skeletal muscle atrophy (p ≤
0.05). Study II attempted to attenuate the effects of cancer cachexia by introducing a
significant hypertrophic stimulus (FO) applied concurrently with tumor development. In
Study II, body mass, skeletal muscle mass, MHC isoform composition, myonuclei/fiber
ratios, CaNα and CaNβ subunit protein content and calcineurin phosphatase activity were
assessed in FO and sham-operated plantaris muscles from healthy and tumor-bearing rats.
Study II: Body Masses
Body masses were obtained once a week for six weeks (Figure 11). When body masses
were determined at termination, animals in the cancer groups, regardless of surgical
intervention, had lost an average of ~6% in adjusted body mass compared to their initial
masses. Animals in the TB-S group lost ~8% in adjusted body mass, while animals in the
TB-FO group lost ~4% in adjusted body mass (Figure 12). One animal in the TB-FO
group gained 7.7% in body mass over six weeks and developed the smallest tumor (0.68
g) in both tumor-bearing groups. However, this animal was not considered an outlier
based on the Grubb’s test and therefore was included in all analyses (Barnett, 1994).
Animals in the vehicle groups gained an ~5% in body mass over the course of six weeks.
Specifically, animals in the Veh-S group gained ~4% in body mass. Animals in the Veh-
FO group gained ~7% in body mass (Figure 12). Surgical intervention (Sham or FO) had
no effect on body mass.
Study II: Plantaris Muscle Masses
Plantaris muscle from animals in the TB-S group were reduced by ~13% compared to the
Veh-S group (p ≤ 0.05) (Table 4). Sham surgeries did not affect plantaris mass, because
left plantaris muscles (i.e., plantaris internal control) were not different in mass from the
right plantaris muscle (i.e., plantaris subjected to FO or sham-surgery).
45
The right/left plantaris ratio of absolute mass is a reliable index of hypertrophy because
previous work has indicated that unilateral FO does not affect the contralateral plantaris
(i.e., left plantaris) (Spangenburg, 2002). Therefore, ratios greater than 1.0 reflect
increased right plantaris mass (i.e., hypertrophy). All other graphs represent data from
the right plantaris muscle only and do not reflect the right/left plantaris ratio.
FO plantaris, regardless of health status (i.e., Veh or TB), increased mass ~34%
compared to internal plantaris controls. Specifically, FO plantaris muscles from animals
in the Veh-FO group increased ~44% compared to internal controls (Figure 13).
Plantaris muscles from animals in the TB-FO group increased ~24% compared to internal
controls. Therefore, FO successfully reversed plantaris atrophy in tumor-bearing animals.
However, plantaris muscles from animals in the TB-FO groups did not hypertrophy to the
same extent as plantaris muscles in the Veh-FO group. Therefore, plantaris muscles from
animals in the TB-FO group appear to have a reduced capacity to hypertrophy compared
to the Veh-FO group.
Study II: Myosin Heavy Chain (MHC) Isoform Expression
Little data exists on the effects of cancer cachexia on skeletal muscle protein content,
specifically the quality of MHC isoforms expressed. Recent data from tumor-bearing rats
has indicated that the diaphragm muscle does not display alterations to MHC isoform
composition (Otis, 2003). This suggests that respiratory dysfunction associated with
cachectic cancer patients is not due to alterations to MHC isoform composition, but are
likely due to increased proteasome activity and resultant diaphragm atrophy. However,
potential alterations to MHC isoform composition in peripheral skeletal muscles, like the
plantaris, might influence contractile physiology (i.e., contractile velocity, maximum
voluntary force outputs) and contribute to muscle fatigue associated with cancer
cachexia. FO plantaris muscle has been shown to stimulate fast-to-slow MHC isoform
transitions. Therefore, FO plantaris muscles might attenuate alterations to MHC in
cachectic, tumor-bearing animals and improve force outputs and muscle endurance.
46
MHC type I
On average, plantaris muscles from animals in the Veh-S group contained ~6% MHC
type I. As expected, FO plantaris from animals in the Veh-FO group had significantly
greater percentages of MHC type I compared to animals in the Veh-S group (Figure 14).
Tumor-bearing animals in the TB-S group displayed reduced percentages of MHC type I
compared to animals in the Veh-S group (p ≤ 0.05). However, FO successfully
attenuated reductions to MHC type I isoform expression. FO plantaris muscles in the
TB-FO group displayed greater percentages of MHC type I compared to plantaris
muscles from animals in the TB-S group (p ≤ 0.05). In fact, MHC type I expression in
plantaris muscles from animals in the TB-FO group was not different from MHC type I
expression in plantaris muscles from animals in the Veh-FO group. Therefore, presence
of a tumor did not appear to inhibit adaptation of MHC isoforms to a hypertrophic
stimulus.
MHC type IIa
On average, plantaris muscles from animals in the Veh-S group contained ~14% MHC
type IIa. Plantaris muscles from tumor-bearing animals in the TB-S group contained
similar percentages of MHC type IIa (~13%).
FO did not affect the percentage of MHC type IIa relative to the total pool of MHC
isoforms. Specifically, plantaris muscles from animals in the Veh-FO group contained
~18% MHC type IIa. Plantaris muscles from animals in the TB-FO group contained
~16% MHC type IIa.
MHC type IIx
On average, plantaris muscles from animals in the Veh-S group contained ~32% MHC
type IIx. Plantaris muscles from tumor-bearing animals in the TB-S group contained
similar percentages of MHC type IIx (~33%).
47
FO did not affect the percentage of MHC type IIx relative to the total pool of MHC
isoforms. Specifically, plantaris muscles from animals in the Veh-FO group contained
~33% MHC type IIx. Plantaris muscles from animals in the TB-FO group contained
~36% MHC type IIx.
MHC type IIb
On average, plantaris muscles from animals in the Veh-S group contained ~49% MHC
type IIb. FO plantaris muscles from animals in the Veh-FO group contained a reduced
percentage (~40%) of MHC type IIb compared to the Veh-S group (p ≤ 0.05).
Presence of tumors did not increase the percentage of MHC type IIb in plantaris muscle.
Specifically, atrophied plantaris muscles from animals in the TB-S group contained
~51% MHC type IIb. However, FO reduced the percentage of MHC type IIb expression
in tumor-bearing animals (p ≤ 0.05). Specifically, plantaris muscles from animals in the
TB-FO group contained ~40% MHC type IIb. In fact, MHC type IIb expression in
plantaris muscles from animals in the TB-FO group was not different from animals in the
Veh-FO group.
Study II: Myonuclei/Fiber Ratios
The addition of new myonuclei to the existing pool, termed myonuclear accretion, helps
to accommodate increased gene transcription and then protein translation during muscle
hypertrophy (see Figure 20 in appendix). In contrast, atrophying fibers might experience
reduced protein translation and increased myonuclear apoptosis that result in reductions
to the myonuclei/fiber ratio. Thus, growth dynamics (i.e., atrophy or hypertrophy) and
the rates of protein translation govern the number of myonuclei that supply a muscle
fiber.
The myonuclei/fiber ratio from plantaris muscles in the TB-S group was ~78% of the
ratio reported in the Veh-S group (p = 0.10) (Figure 15).
48
FO increased the myonuclei/fiber ratio compared to sham-operated controls (p ≤ 0.05).
For instance, animals in the Veh-FO group had myofiber/nuclei ratios ~34% higher than
animals in the Veh-S group.
FO also reversed plantaris muscle atrophy in tumor-bearing animals. This increase in
mass may partially be explained by increased myonuclei/fiber ratios of animals in the
TB-FO group compared to animals in the TB-S group (p ≤ 0.05). Tumor-bearing animals
in the TB-FO group had myofiber/nuclei ratios ~33% higher than animals in the TB-S
group.
Study II: Calcineurin Subunit Expressions
Calcineurin-mediated dephosphorylation of NFATc1 and MEF2 may signal downstream
regulation of skeletal muscle fiber size and protein phenotype expression (Chin, 1998;
Wu, 2000). The endogenous, functional calcineurin enzyme consists of a catalytic
subunit, CaNα, and a regulatory subunit, CaNβ . However, subunit protein content has
not been described in FO skeletal muscle. Furthermore, the influence of an atrophic
stimulus (i.e., cancer cachexia) on subunit protein content has not been evaluated to the
author’s knowledge. Alterations to CaNα and/or CaNβ protein content might forecast
changes to functional calcineurin activity (Mitchell, 2002) and alter growth and/or fiber
type-specific protein expression.
Calcineurin-α (CaNα ) expression
FO surgeries did not result in significant changes to CaNα protein content in either group
(Figure 16). For instance, CaNα protein content from the Veh-S group were ~87% of the
levels expressed in the Veh-FO group. Furthermore, CaNα protein content from the TB-
S group were ~84% of the levels expressed in the TB-FO group.
49
Calcineurin-β (CaNβ ) expression
Presence of a tumor had no affect on CaNβ protein content (Figure 17). CaNβ protein
content in plantaris muscles from the TB-S group were ~90% of the protein content from
the Veh-S group.
Likewise, FO had no effect on CaNβ protein content. CaNβ protein content in plantaris
muscles from the Veh-FO group were ~80% of the protein content from the TB-FO
group.
Study II: Calcineurin Phosphatase Activities
Calcineurin phosphatase activities were initially normalized to total protein (total
phosphatase activity). Specific activities were normalized to CaNα protein content.
Neither total nor specific calcineurin activities were different the experimental groups
(Figure 18). Plantaris muscles from animals in the Veh-FO group reported specific
calcineurin activities ~ 90% of the activities from the Veh-S group. Plantaris muscles
from animals in the TB-FO group reported activities ~ 97% of the activities from the TB-
S group. Furthermore, presence of a tumor had no affect on specific calcineurin activity.
50
Time for tumor development (weeks)
Control 2 4 6
Body Mass (g) 166.2 ± 4.2 151.6 ± 2.6 150.4 ± 3.8 156.8 ± 1.5
Tumor Mass (g) - - 6.5 ± 0.5 12.9 ± 2.5
Adjusted Body Mass (g) 166.2 ± 4.2 151.6 ± 2.6 143.9 ± 3.1 * 143.9 ± 2.1 *
Table 2. Body masses from tumor-bearing rats at 2, 4 or 6 weeks of tumor growth. Tumors were not palpable until after 2 weeks of growth. Adjusted body mass has been corrected for tumor mass. * Adjusted body masses were significantly reduced at 4 and 6 weeks of tumor growth compared to controls (p ≤ 0.05). Data are presented as means ± standard error.
51
Tumor mass (g)
0 5 10 15 20 25 30
% c
hang
e in
adj
uste
d bo
dy m
ass
-20
-15
-10
-5
0
5
10
Figure 7. Correlation between tumor mass and percent change in adjusted body mass. The presence and severity of cachexia as determined by changes to body mass over four and six weeks of tumor development were moderately correlated to tumor mass (r = -0.81; r2 = 0.66).
52
Time (Days)
0 10 20 30 40
Foo
d ea
ten
(g)
12
14
16
18
20
22
24
26VehicleTB
Figure 8. Eating habits of healthy and tumor-bearing rats over 6 weeks. The extreme weight loss associated with cancer cachexia could not be explained by changes to food consumption (i.e., anorexia). Food intake was monitored by providing a premeasured amount of food and weighing the remaining food, including any visible food scattered in the cage.
53
Skeletal Muscles
Soleus Gastroc Plantaris
Abs
olut
e m
ass
(mg)
0
200
400
600
800
1000
Control Two weeksFour weeks Six weeks
Figure 9. Plantar flexor masses from tumor-bearing rats over time. Gastrocnemius and plantaris absolute muscle masses were significantly decreased only after 6 weeks of tumor development. Soleus muscle mass was unchanged at each time point. Gastroc, gastrocnemius; * Significantly different from healthy controls (p ≤ 0.05).
*
*
54
Skeletal Muscles
TA EDL Diaphragm
Abs
olut
e m
ass
(mg)
0
100
200
300
400
500
Control Two weeksFour weeksSix weeks
Figure 10. Dorsi flexor and diaphragm masses from tumor-bearing rats over time. Muscles from the dorsi flexor group (TA and EDL) and the diaphragm were significantly decreased only after 6 weeks of tumor development. TA, Tibialis Anterior; EDL, Extensor Digitorum Longus; * Significantly different from healthy controls (p ≤ 0.05).
*
*
*
55
Time after MH-7777 cell implantation
Control 2 weeks 4 weeks 6 weeks
Soleus 0.08 ± 0.01 0.14 ± 0.02 0.10 ± 0.01 0.09 ± 0.03
Diaphragm 0.07 ± 0.01 0.17 ± 0.02 * 0.15 ± 0.04 * 0.10 ± 0.01 *
Plantaris 0.07 ± 0.01 0.16 ± 0.03 * 0.20 ± 0.03 * 0.12 ± 0.02 *
Table 3. Maximal proteasome activity. Proteasome activity of diaphragm and plantaris muscles was significantly higher than controls after 2, 4 and 6 weeks of tumor growth. However, proteasome activity of the soleus was comparable to controls at all time points. Values (AU/min*mg-1 protein) are represented as means ± standard error. * Significantly different from controls. (p ≤ 0.05).
56
Time (weeks)
0 1 2 3 4 5 6
Abs
olut
e ch
ange
from
initi
al b
ody
mas
s (g
)
-6
-4
-2
0
2
4
6
8
10
12
14
Veh-S TB-S Veh-FO TB-FO
Figure 11. Absolute changes to body mass over 6 weeks of tumor development. Tumor-bearing rats lost adjusted body mass compared to animals in the vehicle group (p ≤ 0.05). Initial body masses were determined at the time of FO or sham surgeries. Standard error bars have been omitted.
57
Vehicle TB
% c
hang
e in
adj
uste
d bo
dy m
ass
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10Sham FO
Figure 12. Percent change from initial body mass over 6 weeks. Morris hepatoma MH-7777 cell implantation causes significant body mass loss after six weeks. Percentages represent changes from initial body mass measured on the day of surgeries (Sham or FO) to adjusted body measured on the day of termination. * Significantly different from vehicle group. (p ≤ 0.05).
*
*
58
Group Left Plantaris mass (mg) Right Plantaris mass (mg) Veh-S 209.8 ± 11.8 214.5 ± 11.2
Veh-FO 169.6 ± 8.3 241.7 ± 7.6 * TB-S 193.0 ± 9.3 # 187.7 ± 10.9
TB-FO 152.8 ± 11.3 # 183.0 ± 14.1* Table 4. Absolute plantaris muscle masses. Unilateral FO significantly increased absolute plantaris mass. The plantaris muscle from the left limb was used as an internal control. This FO model does not hypertrophy the left plantaris and serves as a true control. The plantaris muscle from the right limb was either overloaded or sham-operated. * Significantly different from internal control. # Significantly different from vehicle group. (p ≤ 0.05).
59
Vehicle TB
Rig
ht/L
eft P
lant
aris
rat
io
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Sham FO
Figure 13. Plantaris mass increases after FO. FO surgeries significantly increased plantaris mass relative to internal controls. FO successfully attenuated plantaris atrophy in the TB-FO group compared to TB-S group. Sham surgeries had no effect on plantaris muscle mass relative to internal plantaris controls. * Significantly different from vehicle group within FO surgeries. # Significantly different from sham surgery within vehicle group. ∇ Significantly different from sham surgery within TB group. (p ≤ 0.05). Right, FO leg; Left, internal control leg; FO, functional overload; TB, tumor-bearing groups.
*,∇
#
60
Veh-S TB-S Veh-FO TB-FO
Per
cent
of t
otal
MH
C p
ool
0
10
20
30
40
50
60
I IIa IIx IIb
Figure 14. Myosin Heavy Chain (MHC) Isoform Distribution. A. Representative MHC isoform distribution following SDS-PAGE. B. FO surgeries increased the percentages of slow, MHC type I fibers and reduced the percentages of fast, MHC type IIb fibers in both Veh and TB groups. Therefore, FO attenuated the slow-to-fast transition evident in plantaris muscles from cachectic, tumor-bearing animals. * Significantly different from vehicle group within sham surgery. # Significantly different from sham surgery within vehicle group. ∇ Significantly different from sham surgery within TB group. (p ≤ 0.05). Veh-S, Vehicle, Sham-operated group; TB-S, Tumor-bearing, Sham-operated group; Veh-FO, Vehicle, functional overload group; TB-FO, Tumor-bearing, functional overload group.
*
# ∇
# ∇
IIaIIx
IIbI
A
B
Veh-S TB-S Veh-FO TB-FO
61
Vehicle TB
Myo
nucl
ear D
ensi
ty (m
yonu
clei
/fibe
r)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 Sham FO
Figure 15. Myonuclear Density. FO significantly increased the myonuclei/fiber ratio in both Veh and TB groups. This increase in the myonuclei/fiber ratio reflects the increase in plantaris muscle mass following FO. * Significantly different from vehicle group within FO. # Significantly different from sham surgery within vehicle group. ∇ Significantly different from sham surgery within TB group. (p ≤ 0.05). FO, functional overload; TB, tumor-bearing groups.
*, ∇
#
62
Vehicle TB
Cal
cine
urin
-alp
ha (A
U)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6Sham FO
Figure 16. Calcineurin-alpha (CaN-α ) subunit expression. A. Representative western blot of the CaN-α subunit. B. CaN-α protein content was not altered by cancer cachexia or by FO. Veh-S, Vehicle, Sham-operated group; TB-S, Tumor-bearing, Sham-operated group; Veh-FO, Vehicle, functional overload group; TB-FO, Tumor-bearing, functional overload group.
Veh-S Veh-FO TB-S TB-FO CaN-α A
B
63
Vehicle TB
Cal
cine
urin
-bet
a (A
U)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Sham FO
Figure 17. Calcineurin-beta (CaN-β ) subunit expression. A. Representative western blot of the CaN-β subunit. B. CaN-β protein content was not altered by cancer cachexia or by FO surgery.
CaN-β A
B
Veh-S Veh-FO TB-S TB-FO
*
64
Vehicle TB
nmol
PO
4 re
leas
ed/µ
g pr
otei
n
(nor
mal
ized
to C
aN-α
pro
tein
con
tent
)
0.00
0.02
0.04
0.06
0.08 Sham FO
Figure 18. Specific calcineurin phosphatase activity. Calcineurin phosphatase activies were determined by a modified Malachite green assay. Phosphate released were normalized to total protein. Specific calcineurin activities were normalized to the catalytic subunit of calcineurin, CaNα. Neither total nor specific calcineurin activity were altered by cancer cachexia or by FO.
65
CHAPTER FIVE - DISCUSSION
66
Introduction
Major Findings
The major findings of this study were:
(1) The Morris hepatoma MH-7777 cell line produces significant skeletal muscle
atrophy after six weeks of tumor growth.
(2) This atrophy is accompanied by a slow-to-fast shift in MHC isoform composition
in the plantaris muscle.
(3) FO successfully reverses the atrophic response in tumor-bearing rats, but plantaris
muscle does not hypertrophy to the same extent as plantaris muscles from healthy
controls.
(4) FO successfully increases the percentage of slow, MHC type I in plantaris
muscles from tumor-bearing rats comparable to a trained muscle.
(5) Calcineurin subunit protein content are unaffected by FO or health status.
Furthermore, calcineurin phosphatase activity does not appear to be involved in
regulating the hypertrophic response to FO plantaris muscles.
Cancer cachexia is a debilitating, paraneoplastic syndrome commonly associated with
late stage malignancy. Cachexia is marked by drastic reduction in body mass, primarily
due to increased degradation of skeletal muscle and mobilization of adipose tissue. It is
estimated that ~25% of cancer-related deaths are due directly to complications arising
from cachexia (Barton, 2001). Cachectic patients often present severe asthenia,
generalized weakness and decreased ability to endure contemporary anti-neoplastic
therapies, which often culminates in decreased survival times (Baracos, 2000; Fearon,
2002).
The purpose of the current study was to test the hypothesis that skeletal muscle atrophy
induced by cancer cachexia could be reversed by introducing a profound hypertrophic
67
stimulus (FO). While strength training provides several benefits to cachectic cancer
patients, including increased tolerance to chemotherapy, little data exists on specific,
intrinsic adaptations to skeletal muscles. Specifically, little is known about potential
alterations to force-velocity characteristics, power output, metabolic properties, Ca2+-
handling dynamics, MHC isoform composition or fatigability. Certainly, other disuse
and/or disease models, including congestive heart failure (Spangenburg, 2002), limb
immobilization (Jänkälä, 1997), spinal cord injury (Jiang, 1991; Talmadge, 1995) and
hindlimb suspension (Bigard, 1997) are accompanied with significant muscle atrophy and
have well documented changes to muscle function and protein expression. For example,
our laboratory has indicated that FO increased plantaris mass and fiber cross-sectional
areas in CHF rats and altered Ca2+-uptake rates and MHC isoform expression to resemble
trained muscles (Spangenburg, 2002). Therefore, strength training may improve various
aspects of skeletal muscle physiology in cachectic cancer patients.
Specific Aims
There were three specific aims:
(1) To identify the effects of FO on plantaris muscle masses and myonuclei/fiber
ratios in tumor-bearing rats compared to tumor-bearing, sham-operated controls;
(2) To identify the effect of FO on MHC isoform composition in tumor-bearing rats
compared to controls; and
(3) To identify the effects of FO on calcineurin subunit protein content and
calcineurin phosphatase activity in tumor-bearing rats compared to controls.
68
Body mass loss and skeletal muscle atrophy associated with cancer cachexia
Cancer cachexia results in severe body wasting, primarily due to increased degradation of
skeletal muscle proteins and mobilization of adipose tissue. Tumor-bearing rats had
reduced body masses compared to healthy controls (p ≤ 0.05). These reductions could
not be explained by altered food consumption (i.e., not induced by anorexia).
Several skeletal muscles from tumor-bearing rats were atrophied compared to controls,
including gastrocnemius, plantaris, TA, EDL and diaphragm (p ≤ 0.05). This systemic
atrophic response suggests that atrophy was not due to physical crushing/mechanical
injuries from the tumor proper. Indeed, several studies indicate skeletal muscle
degradation in tumor-bearing animals is a systemic response initiated by circulating
factors released from the tumor proper or infiltrating immune cells, including IL-1, TNF-
α and IFN-γ (Argiles, 1997; Lorite, 2001; Fearon 2002). The most recently discovered
factor involved in cachexia- induced skeletal muscle degradation is proteolysis- inducing
factor (PIF) (Lorite, 1998). PIF, a 24-kDa sulphated glycoprotein, has been isolated from
the urine of cancer patients (Baracos, 2000) and from tumor cells in vitro (Todorov,
1997). Purified PIF introduced into C2C12 myoblasts drastically increases skeletal muscle
degradation (~1.5 fold greater than controls) in a dose-related matter with a maximal
effect between 2-4 nM (Lorite, 2001). This effect of elevated skeletal muscle breakdown
is attenuated with addition of monoclonal antibody raised against PIF. Therapeutically
blocking PIF with eicosapentaenoic acid (EPA) attenuates the development of cachexia
and allows for the accumulation of lean body mass (Hussey, 1999; Tisdale, 2001).
This study indicated time-dependent increases in ATP-independent proteasome activity
in plantaris and diaphragm muscles from tumor-bearing rats compared to healthy
controls. For instance, no change to proteasome activity in the plantaris or diaphragm
was apparent after two weeks of tumor development. However, proteasome activity in
these muscles was increased after four weeks and remained elevated after six weeks of
tumor growth (p ≤ 0.05). Therefore, it is possible that proteasome activity was
consistently increased over the final two weeks of tumor growth. Two weeks of elevated
proteasome activity could produce significant muscle atrophy, particularly in light of
69
reduced muscle protein synthesis often associated with cancer cachexia (Baracos, 2000).
In fact, significant muscle atrophy was apparent only after six weeks of tumor growth.
Overall, these data support the notion that ATP-dependent, ubiquitin-proteasome
activation is central to skeletal muscle atrophy in late stage malignancy (Llovera, 1994;
Lorite, 1998; Schwartz, 1999).
Interestingly, soleus muscle, the primary load-bearing muscle of the plantar flexor group,
did not display increased proteasome activity or significant atrophy at any time following
MH-7777 cell implantation. Yet, Diffee and colleagues (2002) reported a ~49%
reduction in soleus mass in mice bearing a murine C-26 adenocarcinoma implanted
subcutaneously between the scapula. In this model, the physical tumor burden was not
supported by the soleus muscle, but rather distributed across musculature of the front
limbs. In the present model, MH-7777 cells were subcutaneously implanted on the dorsal
side above the hip. In this location, the physical tumor burden potentially preserved
soleus muscle mass possibly by providing a large load and significant hypertrophic
stimulus. Interestingly, localization of a load over the hindlimbs of mice has been shown
to hypertrophy the soleus muscle without affecting other muscles of the lower leg
(Awede, 1999). Lead weights equivalent to one-third body weight inserted underneath
the skin of the lower part of the back increased soleus masses ~17% compared to
controls, but did not affect EDL masses. Tumor masses from the current study averaged
~8% of total body mass which might have been sufficient to maintain, but not increase
soleus mass. The maintenance of soleus mass indicates that increased weight bearing
(i.e., strength training) may prevent skeletal muscle atrophy in cachectic, tumor-bearing
animals. On the other hand, chronic activity may attenuate atrophic responses
independent of load.
70
Hypertrophic response of atrophied muscles from cachectic, tumor-bearing rats
Neuromuscular activity - defined as the combination of neural activity and mechanical
loading (Edgerton, 2002) - in FO plantaris muscles increases to support body mass
following the loss of the synergistic gastrocnemius (Roy, 1982, 1991; Dunn, 2001).
Additionally, FO plantaris muscle responds by increasing protein synthesis rates
(Goldspink, 1991) resulting in larger cross-sectional areas and muscle masses (Baldwin,
1982). Several laboratories have reported ~30-100% increases in plantaris muscle mass
following various durations of compensatory hypertrophy (for review see Lowe, 2002).
In fact, our laboratory has shown ~30% increase in plantaris muscle mass nine weeks
after unilateral FO compared to controls (Spangenburg, 2002). In the current study a
~44% increase in plantaris muscle mass was observed six weeks after unilateral FO
compared to controls.
FO plantaris muscles from tumor-bearing rats increased in mass by ~23% compared to
controls. Therefore, FO reversed plantaris muscle atrophy associated with cancer
cachexia. Increased plantaris muscle mass and the preservation of soleus muscle mass
support the notion that increased weight bearing can benefit atrophied skeletal muscles
from cachectic, tumor-bearing animals.
Yet, plantaris muscles from tumor-bearing rats appear to have a reduced capacity to
hypertrophy compared to FO controls. This blunted hypertrophic response is intriguing
when one considers the muscle was not only supporting body weight, but also supporting
the physical tumor mass. This may be partially explained by the different nitrogen
balance states of tumor-bearing rats and healthy controls. Cancer is frequently associated
with a negative nitrogen balance due to increased skeletal muscle protein degradation and
suppressed muscle protein synthesis rates (Baracos, 2000). Conversely, hypertrophying
muscles are associated with a positive nitrogen balance due to enhanced synthesis of new
proteins, whilst protein catabolism may be either decreased or increased (Goldspink,
1991). When the two treatments are applied in concert (i.e., FO and cancer), it is likely
that hypertrophying muscles from tumor-bearing animals exist in a less positive nitrogen
71
balance state, which results in less contractile protein deposition and accumulation
compared to healthy animals.
It is unclear if FO has a direct effect on the circulating cytokine levels associated with
cancer cachexia and increased skeletal muscle degradation. Interestingly, rats trained to
run on a treadmill display reduced plasma TNF-α levels in response to
lipopolysaccharide challenges (Bagby, 1994). This response was attributed to a reduced
ability of macrophages to release inflammatory factors during subsequent challenges.
Alternatively, exercise might acutely modulate the response of stress hormones and
catecholamines that effectively suppress TNF-α levels (Beutler, 1986).
Myosin heavy chain isoform composition
It is generally accepted that MHC isoform composition is regulated by the amount and
pattern of electrical activity a muscle receives (for review see Roy, 1991). For example,
reduced neuromuscular activity due to spinal cord transection (Talmadge, 1995) or spinal
cord isolation (Jiang, 1991) increased the percentage of fast fiber-like protein phenotypes.
In contrast, increased neuromuscular activation due to tonic, low frequency stimulation
(Pette, 1985) or FO (Dunn, 1999) induces the slow fiber- like protein phenotype. In the
current study, FO plantaris muscles from healthy rats displayed an increased percentage
of MHC type I and a decreased percentage of MHC type IIb, characteristic of the fast-to-
slow transition commonly observed in endurance-trained muscle.
It is unlikely that the tumor location interfered with the conductance of electrical activity
from the spinal cord to the plantaris muscle (i.e., mechanical pressure injury to sciatic
nerve). This observation is supported by the fact that tumor-bearing rats appeared pain-
free and ambulated normally. However, the pattern of neuromuscular activity does not
solely regulate MHC isoform protein expression. Expression patterns of MHC isoform
types have been linked to mechanisms not associated with altered neuromuscular activity,
including hormonal status. For instance, altered levels of circulating growth hormone
(Laughna, 1994), thyroid hormone (Caizzo, 1991) or TNF-α (Dalla Libera, 2001) have
72
been shown to induce slow-to-fast MHC isoform transitions. Our laboratory has shown
increased MHC type IIx isoform expression in plantaris muscle from CHF rats compared
to controls (Spangenburg, 2002), potentially due to increased circulating TNF-α levels
(Dalla Libera, 2001). In fact, TNF-α levels are increased in cachectic cancer patients
(Moldawer, 1997; Tisdale, 2001) and are significantly elevated in SCID mice implanted
with Morris hepatoma MH-7777 cells. Therefore, a fast MHC profile in the plantaris
muscle from tumor-bearing rats in the current study is not surprising.
FO plantaris from tumor-bearing animals retain the capacity to establish a slow MHC
fiber profile characteristic of trained muscle. Although myofibrillar proteins have
relatively long half- lives (~7-10 days) (Baldwin, 2002), these half- lives are significantly
reduced if myofibrils are disassembled from the contractile complex (Samarel, 1992).
Myofibrils can be disassembled from the contractile complex by a Ca2+-dependent
calpain mechanism, which facilitate myosin and actin protein degradation by the ATP-
dependent, ubiquitin-proteasome system (Attaix, 1998). Muscles from tumor-bearing
rats display increased calpain activity levels, decreased calpastatin activity levels (an
endogenous inhibitor of calpain) and an increased calpain/calpastatin ratio (Temparis,
1994; Baracos, 1995; Costelli, 2001). Additionally, cachectic cancer patients display
increased levels of ubiquitin-protein conjugates (Llovera, 1994; Lorite, 1998; Schwartz,
1999) and increased ubiquitin and proteasome subunit mRNA levels (Baracos, 1995).
Therefore, the increased protein turnover rates associated with cancer cachexia provide a
mechanism for contractile proteins to adapt to novel physiological demands imposed on
the muscle. Although cancer cachexia is associated with increased protein degradation
and decreased protein synthesis, FO plantaris from tumor-bearing rats retain the capacity
to synthesize MHC protein isoforms (i.e., MHC type I) at comparable levels evident in
healthy controls. Therefore, strength training might provide relative benefits to cancer
patients, including increased tolerance to conventional therapies and improved quality of
life.
73
Influence of myonuclear apoptosis and accretion
Each myonucleus within a fiber is responsible for maintenance of a specific and limited
cytoplasmic region, referred to as the “DNA unit” (Cheek, 1985) or “myonuclear
domain” (Hall, 1989). To maintain the myonuclear domain, myonuclear numbers are
reduced in concert with reduced cytoplasmic volume in a variety of atrophy models. For
example, myonuclear number is reduced during hindlimb suspension (Allen, 1997),
spaceflight (Allen, 1996) and spinal cord injury (Allen, 1995), which might partially
explain the decreased fiber size. Surprisingly, myonuclei/fiber ratios from atrophied
plantaris muscles in tumor-bearing rats were not significantly different from muscle in
healthy controls. It is possible that skeletal muscle degradation associated with cancer
cachexia occurs independently from myonuclear apoptosis and fiber degeneration. This
possibility seems unlikely because maintenance of myonuclear domains would require
loss of myonuclei as cross-sectional areas and cytoplasmic volumes are decreased.
Therefore, while not statistically significant (p = 0.10), the ~22% reduction in the
myonuclei/fiber ratio in tumor-bearing rats compared to healthy rats might sufficiently
maintain this theoretical myonuclear domain. Unfortunately, the fixation procedures
used in the immunohistochemical analysis of dystrophin to identify the sarcolemma
crosslink proteins and did not allow for precise measurements of fiber cross-sectional
area or cytoplasmic volume. Therefore, the current study was unable to accurately define
myonuclear domains. However, plantaris wet masses from tumor-bearing rats were
reduced by ~13% compared to healthy rats. These similar reductions to the
myonuclei/fiber ratio and plantaris wet mass in tumor-bearing rats might normalize the
myonuclear domain within its theoretical bounds.
There does appear to be a fiber-type specific difference in myonuclear domain size. In
general, fibers from predominantly slow, postural rat muscles have a greater myonuclear
density and hence smaller myonuclear compared to predominantly fast muscles
(Burleigh, 1977; Atherton, 1980). While little is known about why this apparent
discrepancy exists, it has been suggested that slow, oxidative fibers require greater
synthesis of metabolic enzymes (Tseng, 1994) or have greater protein turnover rates
compared to fast muscle (Booth, 1991). Because plantaris muscles from tumor-bearing
74
rats expressed less MHC type I, it is possible that the myonuclei/fiber ratio decreased to
supply proteins commonly associated with a faster MHC profile. This “matching” of
myonuclei/fiber ratio to MHC profile is also supported by the increased myonuclei/fiber
ratio and slower MHC profile of plantaris muscle following six weeks of FO in control
rats.
A growing body of evidence suggests that the addition of new myonuclei contributed by
satellite cell activation and fusion to adult fibers is required to maintain some finite ratio
between total nuclei count and cytoplasmic volume during muscle hypertrophy
(Rosenblatt, 1994; Allen, 1995, 1997). For example, myonuclear number is increased
during FO (Roy, 1985; Allen, 1995), weight training (Kadi, 2000) and in vitro IGF-1
administration (Vandenburgh, 1991). FO plantaris, regardless of health status, was
accompanied by significantly higher myonuclei/fiber ratios compared to sham-operated
rats. However, the myonuclei/fiber ratio in FO plantaris from tumor-bearing rats was
~77% of values from FO controls. This can be partially explained by the extent of
hypertrophy. FO plantaris from tumor-bearing rats only increased ~23% compared to
~44% increase in plantaris mass from healthy rats. Therefore, the increased
myonuclei/fiber ratio and larger FO plantaris masses, regardless of health status, might
normalize myonuclear domains within its theoretical bounds. Nevertheless, cancer
cachexia does not appear to completely prevent the addition of new myonuclei required
to support increased protein synthesis and cell growth.
Calcineurin activity is not associated with plantaris hypertrophy
One of the main goals of this study was to identify the calcineurin pathway as a potential
mechanism responsible for the hypertrophic response, increased myonuclei/fiber ratio
and the transition toward a slower MHC profile in FO plantaris muscles from tumor-
bearing rats. Recently, calcineurin has been identified as a critical factor regulating
skeletal muscle growth and fiber-type dependent gene expression (Chin, 1998; Wu, 2000;
Olson, 2000; Otis, 2001). In fact, we have previously shown that plantaris muscles from
transgenic mice expressing a constitutively active form of calcineurin display
75
significantly greater percentages of slow MHC, slow TnI and SERCA2a isoforms and
increased succinate dehydrogenase activity. However, these same fibers do not display
increased cross-sectional areas compared to control mice (Otis, 2002).
Calcineurin is a heterodimer, comprised of catalytic (CaNα) and regulatory (CaNβ)
subunits (Rusnak, 2000). At present, very little data exist on calcineurin subunit protein
content in FO plantaris muscles (Dunn, 1999). Certainly, no data exist on calcineurin
subunit protein content in atrophic plantaris muscles from tumor-bearing rats. Because
calcineurin activity has been associated with regulation of skeletal muscle mass and slow
fiber- like protein expression, it was hypothesized that atrophied plantaris muscles from
tumor-bearing animals might display reduced subunit protein content and/or reduced
calcineurin activity. However, protein content of both subunits and calcineurin activity
were unaffected by cancer or by FO.
In fact, several studies support the notion that there is no significant increase in
calcineurin activity during muscle hypertrophy (Dunn, 1999; Bodine, 2001). Yet, there
does appear to be stages of skeletal muscle hypertrophy that are calcineurin-dependent.
For example, CsA administration does not prevent hypertrophy induced by two weeks of
FO. However, CsA administration does prevent hypertrophy after four weeks of FO
(Dunn, 1999). This late effect of calcineurin inhibition would either be consistent with a
second form of late hypertrophy, which is indeed calcineurin-dependent, or with a non-
specific toxicity caused by CsA (Bodine, 2001). From the current study, it is unclear if
calcineurin plays a vital role in regulating the growth response after six weeks of
compensatory hypertrophy. However, because calcineurin subunit protein content and
phosphatase activity were unchanged in any group, it is unlikely that calcineurin explains
the hypertrophic response in FO plantaris from healthy rats or the blunted hypertrophic
response in FO plantaris from tumor-bearing rats. This notion is further supported by the
fact that transgenic mice overexpressing a constitutively active form of calcineurin do not
report increased fiber cross-sectional areas (Dunn, 2000; Naya, 2000; Otis, 2002).
76
Furthermore, studies that administer CsA as a calcineurin antagonist are controversial.
Muscle mitochondrial respiration is decreased due to suppression of mitochondrial
electron chain capacity following 2 weeks of CsA administration (Hokanson, 1995;
Mercier, 1995). CsA also reduced IGF-1 signaling (Grounds, 2002). Finally, prolonged
CsA treatment has been associated with cardiovascular dysfunction, including
cardiotoxicity (Bellamy, 1995), altered characteristics of Ca2+-release channel in cardiac
sarcoplasmic reticulum (Park, 1999), reduced capillary density (Breill, 1999) and reduced
systolic function (Kingma 1991). Our laboratory has shown that reductions to cardiac
function following myocardial infarction altered skeletal muscle MHC composition
(Spangenburg, 2002). Thus, changes in muscle gene expression associated with
calcineurin in animals treated with CsA may be a secondary result of its side effects on
various mechanisms intrinsic and extrinsic to skeletal muscle.
Conclusions
The major finding of this study was that skeletal muscle retains its ability to adapt to
increased loading in the presence of cancer cachexia. This was evidenced by the muscle
hypertrophy and the slow MHC profile in FO plantaris muscle from tumor-bearing
animals. In addition, FO in tumor-bearing animals increased myonuclei/fiber ratios in
plantaris. Collectively, these data suggest that cancer cachexia does not prevent skeletal
muscle adaptations resulting from increased activity and load bearing.
Calcineurin subunit protein content and phosphatase activity appeared to be unaffected
by FO or cancer. Therefore, calcineurin phosphatase activity did not appear to be
associated with the growth response in FO plantaris muscles from healthy or tumor-
bearing animals.
77
Future Directions
Although cancer cachexia results in significant muscle mass loss and generalized
weakness, little is known about the effects on other aspects of muscle function such as
force-velocity characteristics, power output, metabolic properties, Ca2+-handling kinetics,
MHC isoform composition and fatigability. Certainly, detailed analysis of these skeletal
muscle physiology parameters is warranted.
While this study failed to associate calcineurin activity with regulating the morphological
and physiological response of hypertrophying plantaris muscles in cachectic, tumor-
bearing rats, several other mechanisms potentially influence skeletal muscle growth,
including IGF-1 (Adams, 2002), myostatin (Lee, 2001) and myoD (Alway, 2002)
pathways. Exploiting these regulatory pathways may enhance satellite cell activation and
provide therapeutic interventions for skeletal muscle wasting associated with cancer
cachexia.
78
CHAPTER SIX - REFERENCES
79
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APPENDIX
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Weekly Body Mass (g)
Animal 0 1 2 3 4 5 6
Total Tumor Mass (g)
Adj. BM (g)
PlantLT (mg)
PlantRT (mg)
VEH-S1 181.9 183.3 182.7 185.6 187.2 189.1 187.6 - 187.6 215 218 VEH-S2 168.2 169.1 170.1 169.7 170.7 170.3 171.9 - 171.9 182 191 VEH-S3 192.1 199.6 200.6 200.9 204.9 204.7 207.4 - 207.4 222 229 VEH-S4 174.0 173.0 173.7 171.6 176.0 172.0 174.6 - 174.6 219 225 VEH-S5 196.0 202.8 201.1 199.7 202.3 200.5 203.2 - 203.2 250 250 VEH-S6 164.0 167.8 170.9 166.8 165.8 166.6 169.0 - 169.0 171 174 TB-S1 172.0 174.3 174.1 167.9 165.5 164.4 166.7 8.99 157.7 170 166 TB-S2 173.2 177.5 172.3 165.7 166.4 170.2 177.1 24.67 152.4 178 181 TB-S3 206.0 213.6 207.5 202.0 199.6 198.4 206.9 14.73 192.2 209 201 TB-S4 177.0 180.0 180.7 170.3 167.7 166.9 170.5 12.91 157.6 179 165 TB-S5 170.0 171.3 171.8 174.1 172.0 167.5 179.5 17.76 161.7 192 178 TB-S6 177.0 179.6 182.5 185.1 181.7 178.8 183.9 8.45 175.5 230 235
VEH-FO1 180.4 182.6 183.4 188.1 181.7 186.8 189.6 - 189.6 152 229 VEH-FO2 175.4 177.2 178.5 181.3 184.2 185.1 188.7 - 188.7 187 266 VEH-FO3 199.3 196.9 200.0 199.7 202.2 214.5 216.2 - 216.2 155 250 VEH-FO4 187.8 187.1 187.6 186.9 187.1 193.5 195.7 - 195.7 152 257 VEH-FO5 153.6 159.3 163.7 170.4 170.4 172.3 165.5 - 165.5 172 218 VEH-FO6 161.4 168.0 170.8 177.7 182.3 183.4 177.8 - 177.8 200 230 TB-FO1 166.0 171.4 177.1 182.2 181.2 182.5 180.5 0.68 179.8 237 235 TB-FO2 152.3 150.7 154.3 147.0 152.8 156.1 156.4 14.3 142.1 120 179 TB-FO3 159.3 162.9 163.2 157.7 157.6 161.3 170.0 10.0 160.0 142 178 TB-FO4 156.9 159.8 165.2 161.8 162.4 164.2 160.3 5.59 154.7 131 174 TB-FO5 155.9 156.3 157.4 153.6 149.4 NA NA NA NA NA NA TB-FO6 146.4 150.5 150.8 153.1 155.3 155.0 142.1 16.6 125.5 134 149
Table 5. Morphological Data. Adj. BM, Final body masses adjusted for tumor mass; LT, internal control; RT, overloaded or sham-operated; NA, Animal died during experimental time frame.; VEH-S, Vehicle- injected, Sham-operated; TB-S, Tumor-bearing, Sham-operated; VEH-FO, Vehicle- injected, Functional overload; TB-FO, Tumor-bearing, Functional overload.
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MHC Isoforms (% of total pool) Animal I IIa IIx IIb
Myo/F CaNa CaNß CaN act.
VEH-S1 6.60 6.60 38.60 48.10 1.83 1.51 0.80 - VEH-S2 3.80 15.40 29.70 51.10 2.00 0.87 0.93 - VEH-S3 6.00 15.10 30.10 48.80 2.12 1.11 1.45 - VEH-S4 10.30 13.30 34.20 42.10 1.88 1.03 0.94 0.02 VEH-S5 5.10 18.40 27.10 49.50 2.02 1.25 0.83 0.03 VEH-S6 6.80 15.00 30.00 48.20 - 0.61 0.54 0.07 TB-S1 0.00 0.00 47.20 52.80 1.38 1.00 1.00 - TB-S2 5.20 16.50 33.50 44.80 1.18 0.41 1.07 - TB-S3 4.20 16.60 29.50 49.60 1.76 0.64 0.96 - TB-S4 3.90 11.30 28.30 56.50 1.29 0.49 0.64 0.12 TB-S5 0.00 16.20 27.90 55.90 2.17 1.12 0.68 0.04 TB-S6 4.30 16.00 30.00 49.70 1.44 0.38 0.64 0.18
VEH-FO1 8.20 18.80 32.60 40.50 2.60 0.91 0.93 0.03 VEH-FO2 9.10 15.00 29.20 46.70 4.21 1.17 1.02 0.08 VEH-FO3 10.10 19.60 27.90 42.40 3.23 0.78 0.72 - VEH-FO4 8.70 19.30 35.50 36.50 2.30 1.04 0.80 0.02 VEH-FO5 8.90 16.50 34.80 39.80 2.82 1.44 0.92 - VEH-FO6 8.90 18.90 33.50 38.60 2.63 2.07 0.75 - TB-FO1 7.90 17.70 33.00 41.40 2.42 0.55 0.85 - TB-FO2 9.20 21.90 34.40 34.50 2.61 1.08 1.08 - TB-FO3 8.00 14.20 34.00 43.80 2.36 0.68 1.09 0.08 TB-FO4 7.40 11.60 39.70 41.20 1.99 0.95 1.15 0.09 TB-FO5 - - - - - - - - TB-FO6 8.30 14.60 39.80 37.40 2.02 0.76 1.18 0.08
Table 6. MHC, myonuclei/fiber ratios, calcineurin subunits and activities data. MHC, Myosin heavy chain; Myo/F, myonuclei/ fiber ratios; CaN, Calcineurin; TB-S, Tumor-bearing, Sham-operated; VEH-FO, Vehicle- injected, Functional overload; TB-FO, Tumor-bearing, Functional overload.
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Figure 19. Raw tracing from ATP-independent ubiquitin, proteasome activity assay. Diaphragm and plantaris muscles from tumor-bearing rats displayed increased ATP-independent proteasome activity relative to healthy controls. These tracings are from plantaris muscles after either 6 weeks of tumor growth (cancer) or vehicle injection (control).
Time (sec) A
U
Control
Cancer
(sec)
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Figure 20. Plantaris stained for dystrophin and counterstained for myonuclei. A. Representation of plantaris muscle cut at 8µm and stained for the sarcolemma protein dystrophin. B. Sections counterstained with Hoechst-33258 to identify myonuclei. 10X magnification.
A
B
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Figure 21. Morris Hepatoma. The Morris Hepatoma MH-7777 cell line produced palpable tumors after ~17-19 days. A. This animal represents a typical tumor growth response after 6 weeks. B. Average tumor dimension were 31-mm in width, 40-mm in length and 22-mm in height after 6 weeks.
A
B
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Figure 22. ATP-dependent-ubiquitin proteasome system. Conjugating enzymes (E1, E2 and E3) poly-ubiquinate the target substrate via ATP-dependent processes. The ubiquinated substrate is degraded into its constituent amino acids by a large, barrel-shaped complex known as the 26S proteasome. These amino acids are available as energy substrates in cancer cachexia.
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CURRICULUM VITAE
JEFFREY S. OTIS, Ph.D. 1706 Boxwood Drive, Apt. C Blacksburg, Virginia 24060 Home: 540-552-4264 Work: 540-231-5375 Email: otis@vt.edu EDUCATION
Virginia Tech, Blacksburg, VA M.S., Muscle Physiology and Biochemistry, 2000 Ph.D., Muscle Physiology and Biochemistry, 2003 The University of Pittsburgh, Pittsburgh, PA Completed 24 Masters- level credits in Clinical Exercise Physiology before transferring to Virginia Tech in 1998 The Pennsylvania State University, University Park, PA B.S., Vertebrate Physiology, 1996 COURSES AND TECHNIQUES
Courses taken include: Advanced Exercise Physiology, Molecular Biology for the Life Sciences, Molecular Biology of the Cell, BioUltrastructure, Skeletal Muscle Function and Exercise, Skeletal Muscle Plasticity, Biochemistry for the Life Sciences Techniques performed include: PCR, Electron Microscopy, Quantitative Histochemistry, Immunohistochemistry, SDS-PAGE, Western Blotting, Cell Culturing (both myoblasts and cancer cells) POSITIONS HELD
Graduate Research Assistant 08/00 to present Virginia Tech Instruct Masters-level and Doctorate- level students on histochemical and biochemical techniques while completing own research objectives Graduate Teaching Assistant 01/99 to 08/00 Virginia Tech
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Courses taught include: Kinesiology, Neuromuscular Adaptations to Exercise, Metabolic Nutrition and Methods for Human Nutritional Assessment POSITIONS HELD
Academic Tutor 08/98 to 12/98 Virginia Tech Instructed student-athletes enrolled in Exercise Physiology and Kinesiology classes Cardiac Rehabilitation Intern 01/98 to 05/98 University of Pittsburgh - Shadyside Hospital Monitored and evaluated cardiac patients during exercise training sessions and testing protocols PROFESSIONAL SOCIETIES
American Physiological Society 1998 to present Society for Experimental Biology and Medicine 1998 to present Southeast American College of Sports Medicine 1999 to 2001 ACADEMIC POSITIONS AND AWARDS
HNFE departmental representative to the Graduate Committee Volunteered student representative to SEACSM for Virginia Tech SEBM Travel Fellowship External Reviewer/Judge (2002) Graduate Research Development Project Grant Awardee (2002) Graduate Student Assembly Travel Grant Awardee (2000, 2002) Raville Scholarship Awardee (2001) SEBM Travel Fellowship Awardee (2001) Hepler Fellowship Awardee (2001)
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ABSTRACTS AND REFEREED PUBLICATIONS
Temporal analysis of proteolytic activity and MHC profile in diaphragm muscles from tumor-bearing rats. J.S. Otis, S.J. Lees, W.R. Huckle and J.H. Williams. FASEB J. 2003 17(4): A438. Effects of activated calcineurin on the metabolic profile of myosin heavy chain-based mouse plantaris fibers. J.S. Otis, F.J. Naya, E.N. Olson and R.J. Talmadge. FASEB J. 2002 16(5): A770. ‡ Effects of calcineurin activation on sarcoplasmic reticulum calcium uptake and release. S.J. Lees, J.S. Otis, J.H. Williams, F.J. Naya, E.N. Olson and R.J. Talmadge. FASEB J. 2002 16(5): A768. Oxidative and glycolytic enzyme activities in mouse plantaris fibers expressing activated calcineurin. J.S. Otis, F.J Naya, E.N. Olson and R.J. Talmadge. 18th annual research symposium of Virginia Tech, 2002. Effects of constitutively active calcineurin on SERCA expression and activity in mouse skeletal muscle. S.J. Lees, J.S. Otis, J.H Williams, F.J. Naya, E.N. Olson and R.J. Talmadge. 18th annual research symposium of Virginia Tech, 2002. Effects of moderate heart failure and functional overload on rat plantaris muscle. E.E. Spangenburg, S.J. Lees, J.S. Otis, R.J. Talmadge, T.I. Musch and J.H. Williams. J. Appl Physiol. 2002 Jan;92(1):18-24. Effects of activated calcineurin on muscle mass and myosin heavy chain isoform expression. R.J. Talmadge, S. Weber, J.S. Otis, F.J. Naya and E.N. Olson. FASEB J. 2001 15(4 part I): A421. † Effects of activated calcineurin on myosin heavy chain (MHC), troponin I (TnI), and sarcoplasmic reticulum Ca2+ATPase (SR-ATPase) isoforms expression. J.S. Otis, R.J. Talmadge, F.J. Naya and E.N. Olson. FASEB J. 2001 15(4 part I): A421. The effects of congestive heart failure and functional overload on rat skeletal muscle. E.E. Spangenburg, J.S. Otis, S.J. Lees, R.J. Talmadge, T.I. Musch and J.H. Williams. The Physiologist 2000 43(4): A364. ‡ Metabolic properties of rat soleus fibers after spinal cord transection. J.S. Otis, R.J. Talmadge, R.R. Roy and V.R. Edgerton. Experimental Biology 2000 (late-breaking), San Diego, California † Awarded Society for Experimental Biology and Medicine 2001 Travel Fellowship. ‡ Manuscript from this study is currently being revised for publication.
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