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.