A COMPARISON OF HEAT SHOCK PROTEIN EXPRESSION IN RAT ... · evaluating muscle Hsp expression in the...

A COMPARISON OF HEAT SHOCK PROTEIN EXPRESSION IN

RAT SKELETAL MUSCLE AFTER LENGTHENING OR

SHORTENING CONTRACTIONS

by

Andrew M. Holwerda

A thesis submitted in conformity with the requirements for the degree of Masters of

Science, Exercise Science

Exercise Science Department

University of Toronto

© Copyright by Andrew M. Holwerda, 2013

Holwerda, A - M.Sc Thesis ii

Abstract

The mechanism and subsequent patterns of Heat shock protein (Hsp) expression in

skeletal muscle specific to contraction type was determined. Rat tibialis anterior (TA) muscle

was forcibly lengthened (LC) or shortened (SC) in 5 sets of 20 repetitions before being removed

at 2, 8, 24, 48, 72, or 168 hours and analyzed for muscle damage and Hsp25 and Hsp72

expression. Isometric peak torque was reduced to 63% and 33% (P<0.001) at 3-minutes after SC

and LC, respectively. Muscle fibre damage appeared at 8h and beyond following LCs, but no

damage was observed after SCs. Hsp25 content in LC muscle increased by 3.1±0.53 fold

(P<0.01) at 48h and remained elevated. Hsp72 content increased by 3.8±0.66 fold at 24h and

remained elevated. Neither Hsp25 nor Hsp72 content was elevated following SCs. Muscle

damage associated with LCs results in a greater Hsp accumulation than SCs and 100 SCs do not

result in increased Hsp content.

Holwerda, A - M.Sc Thesis iii

Acknowledgements

I would like to extend gratitude to my supervisor, Marius Locke, for giving me the

opportunity to complete this project. His guidance and patience helped me navigate through the

crests and troughs of completing such a research project. Thank you for your help and support!

I am also appreciative for my group of friends. I consider them to have been crucial to

the completion of this thesis. Thank you mostly for helping me unwind with laugher when I was

stressed out, and also for frequently providing me with a couch to sleep on when I did not want

to go all the way home for the night (or weekend)!

Lastly, and most importantly, thank you to my family for their continual support and

encouragement. They helped me stay positive and focused even through numerous long and

frustrating days (or weeks) in the lab. Thank you for listening and attempting to follow when I

would go on and on about heat shock proteins, western blotting and rat muscle!

Holwerda, A - M.Sc Thesis iv

Table of Contents

Abstract .......................................................................................................................................... ii

Acknowledgements ...................................................................................................................... iii

Table of Contents ......................................................................................................................... iv

List of Abbreviations ................................................................................................................... vi

1. Introduction ............................................................................................................................... 1

2. Review of Literature ................................................................................................................. 2 2.1 Skeletal Muscle Contraction and Adaptation ................................................................................ 2

2.2.1 Causes of Muscle Damage .......................................................................................................... 4 2.2.2 Changes in Muscle Function ....................................................................................................... 4 2.2.3 Biochemical Changes .................................................................................................................. 8 2.2.4 Fiber-type differences ................................................................................................................. 8 3.3.5 Symptoms of muscle damage ...................................................................................................... 9 3.3.6 The Repeated Bout Effect ........................................................................................................... 9 3.4.1 Hsp families and cellular functions ........................................................................................... 13

Hsp25 ............................................................................................................................................................ 16 Hsp70 ............................................................................................................................................................ 16 eHsp70........................................................................................................................................................... 17

3.4.2 Intracellular Molecular Functions of Hsps ................................................................................ 18 Intracellular Binding of Hsps ........................................................................................................................ 18 Regulation of the Cellular Stress Response ................................................................................................... 19

3.4.3 Hsps and exercise ...................................................................................................................... 20 Endurance Exercise ....................................................................................................................................... 20 Resistance exercise ........................................................................................................................................ 22 Hsps relating to the Repeated Bout Effect ..................................................................................................... 22

3.4.4 The influence of Hsps in muscle growth and loss ..................................................................... 23 Hsps and Muscle Hypertrophy ...................................................................................................................... 23 Hsps Preventing Muscle Atrophy .................................................................................................................. 24

3.4.5 Enhancing Athletic Performance ............................................................................................... 24 3.4.6 Cross-over effects of Hsp induction .......................................................................................... 25

4. Rationale .................................................................................................................................. 25

5. Objectives................................................................................................................................. 27

6. Hypotheses ............................................................................................................................... 27

7. Methods & Materials .............................................................................................................. 27 Animals. ........................................................................................................................................................ 27 Study Design ................................................................................................................................................. 28 Stimulation protocol ...................................................................................................................................... 30 Tissue Collection ........................................................................................................................................... 33 Fibre Morphology ......................................................................................................................................... 33 Statistics ........................................................................................................................................................ 35

8. Results ...................................................................................................................................... 35

9. Discussion................................................................................................................................. 48

10. Conclusion ............................................................................................................................. 54

Holwerda, A - M.Sc Thesis v

11. References .............................................................................................................................. 56

Appendix 1 – Laboratory Protocols .......................................................................................... 68 Analysis Protocol 1: Tissue Preparation and Sectioning Skeletal Muscle .................................................... 68 Analysis Protocol 2: Hematoxylin and Eosin Staining .................................................................................. 71 Analysis Protocol 3: Determination of Protein concentration – Lowry Assay .............................................. 72 Analysis Protocol 4: SDS-PAGE and Western Blotting ............................................................................... 73

Appendix 2 – Compiled Raw Data ............................................................................................ 75

Holwerda, A - M.Sc Thesis vi

List of Abbreviations

% Percent

° Degrees

°C Degrees Celsius

< Less than

> Greater than

ANOVA Analysis of Variance

APS Ammonium Persulfate

AST Aspartate Aminotransferase

ATP Adenosine Triphosphate

BSA Bovine Serum Albumin

Ca2+

Calcium

CK Creatine Kinase

CON Control

CSA Cross Sectional Area

CuSO4 Copper Sulfate

DAMP Damage-associated Molecular Pattern

dd Double Distilled

DNA Deoxyribonucleic Acid

DOMS Delayed Onset Muscle Soreness

DPI Dots per Inch

E-C Excitation-Contraction

EDL Extensor Digitorum Longus

EE Endurance Exercise

eHsp Extracellular Heat Shock Protein

EMG Electromyograph

g Grams

G Gauge

g-cm Gram Centimetres

h Hours

H&E Hemotoxylin & Eosin Stain

H2O Water

HRP Horse Radish Peroxidase

Hsc Heat Shock Cognate

HSE Heat Shock Element

HSF1 Heat Shock Factor

Hsp Heat Shock Protein

Hsp25 Heat Shock Protein 25

Hsp70 Heat Shock Protein 70

Hz Hertz

IL-1β/6/12 Interleukin

IκB Inhibitor of κB

JNK c-Jun N-terminal kinases

K+ Potassium

Holwerda, A - M.Sc Thesis vii

kDa Kilodalton

L Litres

LC Lengthening Contractions

LDH Lactate Dehydrogenase

mg Milligrams

min Minutes

mL Millilitres

mRNA messenger RNA

Na-T Sodium Tartrate

Na+ Sodium

Na2CO3 Sodium Carbonate

NaOH Sodium Hydroxide

NF-κB Nuclear Factor-kappaB

NFSM Non-Fat Skim Milk powder

nm Nanometers

O2 Oxygen

OCT Optimal Cutting Temperature

RBE Repeated Bout Effect

RE Resistance Exercise

RNA Ribonucleic Acid

ROM Range of Motion

ROS Reactive Oxygen Species

S Seconds

SC Shortening Contractions

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis

SEM Standard Error of the Mean

TA Tibialis Anterior

TBS Tris Buffered Saline

TLR2/4 Toll-like Receptor

TNF-α Tumor Necrosis Factor- alpha

TTBS Tris Buffered Saline +Tween

V Volts

VO2 Volume of Oxygen consumed

μg Micrograms

μm Micrometers

Holwerda, A - M.Sc Thesis 1

1. Introduction

Mammalian skeletal muscle has a remarkable capacity to recondition and adapt following

loaded contractions. When a load is applied to contracted muscle the force generated by the

muscle usually overcomes the load and a shortening or concentric contraction occurs. Repeated

shortening contractions, as performed during daily activities or exercise, characteristically result

in muscle fatigue in the form of decreased ability to generate force. In contrast to shortening

contractions, if the load applied to a contracted muscle exceeds the force generated by the

muscle, the muscle lengthens and an eccentric contraction occurs. Repeated lengthening

contractions, performed during resistance exercise or downhill running, can damage structural

components of muscle and impair contractile function. The associated muscle recovery and

adaptation to specific contraction-types is of interest based on the potential to optimally

recondition muscle for increased strength and function.

All cells, including muscle fibres, induce the cellular stress response after being exposed

to non-lethal stressors. A key molecular component of this protective adaptation are heat shock

proteins (Hsps), which are rapidly synthesized and are thought to preserve functional and

structural proteins from denaturation and/or aggregation. In muscle, Hsps are synthesized after

both damaging and non-damaging types of exercise and may enhance muscle adaptation while

providing protection against future damage. Thus, understanding post-contractile mechanisms of

Hsp expression in muscle may allow for optimization of muscle adaptation ultimately enhancing

overall health.

Treadmill running has typically been used to study post-contraction skeletal muscle Hsp

expression in human and rodent subjects. Level or incline running is often used as a model to

study repeated non-damaging contractions and declined running is often used for damaging


contractions. It is possible that performing any form of treadmill running may induce metabolic

stress or increase body temperature or other stressors possibly introducing confounding factors of

Hsp induction. Thus, a more isolated model of skeletal muscle contraction should be used to

study the influence of muscle contraction-type and the subsequent patterns of Hsp expression in

muscle.

The experiment in this thesis sought to describe the temporal pattern of the intramuscular

expression of two protective Hsps for 7-days after a controlled bout of either shortening or

lengthening muscle contractions. The separate contraction-types were stimulated in rat tibialis

anterior muscle in an intermittent bout of 100 contractions. Furthermore, the shortening and

lengthening contractions were matched for contraction velocity and mechanical work to isolate

the variable of contraction-induced damage. The experiment resulted in an in vivo model

evaluating muscle Hsp expression in the post-contraction recovery phase.

2. Review of Literature

2.1 Skeletal Muscle Contraction and Adaptation

Although the term “contraction” typically refers to the action of something decreasing in

size, with respect to muscle, “contraction” implies that muscle has received an action potential

from the central nervous system and is in the process of generating tensile force. A complete

muscle contraction can be separated into three types of contractions: eccentric, isometric and

concentric.

Muscle tensile force is generated by sarcomeres; large lattice structure of parallel inter-

digitated myosin and actin (myofibrillar) proteins. During every type of contraction, numerous

myosin heads will bind to sites on adjacent actin proteins, forming cross-bridges. Once a cross-

bridge is formed, the myofibrillar proteins shift to shorten the muscle fibre, which generates

muscular force. If the external load exceeds the muscular force, then the muscle will be forcibly


lengthened while contracted, which is referred to as an eccentric contraction. During a

lengthening contraction, the myosin heads bound to the actin might be stretched or pulled apart

as the muscle lengthens. Performing lengthening contractions can result in muscle fibre damage

and compensatory adaptations, such as increased intracellular Hsp expression, which serve to

protect from further damage after subsequent lengthening contractions.

Alternatively, if the muscular force overcomes the external load during contraction, the

muscle fibre will shorten, and a concentric contraction will occur. In contrast to lengthening

contractions where cross-bridges are pulled apart, shortening contractions require rapid cross-

bridge cycling, which drastically increases demand for energy-dense adenosine triphosphate

(ATP) molecules. Producing sufficient quantities of ATP within the muscle requires elevated

mitochondrial or glycolytic activity, which disturbs metabolic homeostasis and often generates

harmful byproducts. Muscle adapts to these perturbations of metabolic homeostasis through

enzyme synthesis, mitochondrial biogenesis and improving energy storage with the intent on

sustaining muscle force during future contractions. Although concentric contractions do not

appear to result in any overt muscle fibre damage, metabolic stress can trigger the cellular stress

response causing increased Hsp expression.

Lastly, an isometric contraction occurs when the muscular force is matched with the

external load and there is no change in fibre length. Isometric contractions are not a focus in the

present thesis; however, they are considered to be similar to shortening contractions as they

mostly generate metabolic stress without muscle damage.

2.2 Muscle Damage


2.2.1 Causes of Muscle Damage

For the purpose of this thesis, skeletal muscle damage is defined as disrupted cellular

structures (myofibrils, sarcolemma, sarcoplasmic reticulum, etc) resulting in a temporary

decrease in contractile function (Nosaka & Clarkson, 1995; Tiidus, 2008). Skeletal muscle

damage can be induced from a variety of events including: crush injury, freezing injury and

myotoxin injection, all of which physically abolish muscle structure. Aside from these modes of

injury, the most common and physiologically relevant manifestation of muscle damage is

induced from contraction. Contraction-induced skeletal muscle damage is sustained from either

unaccustomed, load-bearing activity such as resistance exercise or from isolated lengthening

contractions (Nosaka et al., 2001; Tiidus, 2008).

2.2.2 Changes in Muscle Function

Muscle damaged from a bout of lengthening contractions will sustain numerous functional

changes. The most immediate and notable change is the significant decrease in peak force,

which some investigators regard as the most useful way to verify inflicted muscle damage

(Brooks et al., 1995; Warren et al., 1999; McHugh, 2003). In general, a larger decrease in

muscle force indicates a more severely damaged muscle and thus a longer recovery period.

Decreases in peak force from contraction-induced damage are typically 25-50%; however, as

little as 5 eccentric contractions at 150% of pre-injury peak force can cause a 15% decrease in

force (Clarkson & Tremblay, 1988; Faulkner et al., 1989; Warren et al., 1993; 2000; Tiidus,

2008). Moreover, evaluation of the different mechanical components of eccentric contractions

(i.e. peak force, initial length, length change, and lengthening velocity) revealed that relative

force produced in the muscle during the damaging contractions affects post-injury muscle

contractile performance the most (Warren et al., 1993; McHugh et al., 2001).


The mechanisms responsible for the reduction in muscle force are the disruption of

excitation-contraction coupling (E-C) and sarcomere disorganization (Morgan & Allen, 1999;

Sam et al., 2000; Proske & Morgan, 2001). Of these two mechanisms, disruption in E-C

coupling is regarded as the primary contributor (Balnave & Allen, 1995; Warren et al., 1999;

Morgan & Allen, 1999; Allen, 2001; Warren et al., 2001; McHugh, 2003) and involves elevated

cytoplasmic Ca2+

concentration and malfunctioning sarcolemma and T-tubules (Morgan, 1990;

Warren et al., 1993; Lynn & Morgan, 1994; Lynn et al., 1998). The lasting elevation in

cytoplasmic Ca2+

concentration arises from impaired sarcoplasmic reticulum Ca2+

sequestering in

combination with permeabilization of the sarcolemma (McNeil & Khakee, 1992; Malm et al.,

1996; Brockett et al., 2001). Some investigators have attempted to connect the elevated

intracellular Ca2+

with the post-contraction rise in passive tension (Balnave & Allen, 1995; Pizza

et al., 1996; Balnave et al., 1997; Ingalls et al., 1998). This notion is disputed by others who

suggest that abnormal resting intracellular Ca2+

concentrations are only responsible for low levels

of post-contraction muscle tension (Proske & Morgan, 2001; Thompson et al., 2001; Whitehead

et al., 2001; Koh, 2002; 2004; Vasilaki et al., 2006; Paulsen et al., 2007; 2009).

Sarcomere disorganization is facilitated by repeated over-extension while the cross-

bridges are formed, forcing the myofilament overlap beyond the functional range. Moreover,

randomly distributed, “weaker” sarcomeres are unaccustomed to handling such large forces, in

which some myofilaments fail to re-interdigitate, resulting in an unusable section of the

sarcomere(Brown & Hill, 1991). As the actin-myosin interactions are forcibly overcome

throughout an eccentric contraction, focused damage likely occurs directly to the myosin heads

and/or the interacting actin-binding site (Enoka, 1996). Damaged sarcomeres commonly result

in z-band streaming, which manifests as a widening or “smudging” of the framing structures, or


z-bands, of sarcomeres. The occurrence of z-band streaming is a hallmark characteristic of

structural fiber damage and is commonly reported in studies concerned with muscle damage

(Fridén et al., 1983; Chiang et al., 1989; Atomi et al., 1991; Fielding et al., 1993; Malm & Yu,

2012). As the myofilament becomes impaired, increasing forces are focused on cytoskeletal

structures, which are normally only responsible for passive tension (Weitzel et al., 1985; Proske

& Morgan, 2001). As such, various cytoskeletal proteins including desmin, which maintains z-

disk and sarcomere organization, may be disrupted from forced lengthening contractions. Once

disrupted, desmin likely escapes through the damaged sarcolemma as indicated by its decreased

intracellular content following damage (Davies et al., 1982; Lieber & Thornell, 1996; Chong et

al., 1998; Fridén & Lieber, 2001). A graphical timeline of the progression of contraction-

induced muscle damage during recovery is presented in Figure 1, which is adopted from Tiidus

(2008).


Figure 1: Progression of Muscle Damage and Presence of Inflammatory Cells – Adapted

from the original figure displayed in Tiidus (2008). The temporal pattern of contraction-

induced muscle fibre damage (% damaged fibres; grey)

0

5

10

15

20

25

0h 2h 6h 1d 2d 3d 4d 5d

Per

cent

Fib

re D

amag

e (%

)

Time post-contraction bout

Muscle Damage


2.2.3 Biochemical Changes

Contraction-induced muscle damage also alters the biochemical homeostasis of the

affected muscle tissue. Aside from elevated cytokine presence (to be described later) and the

previously described disruption in Ca2+

flux, significant alterations in glucose metabolism and

cytoplasmic protein “release” can result from muscle damage. Studies have reported impaired

glucose uptake and glycogen synthesis in damaged muscle apparent in the sustained elevations in

insulin (King et al., 1993; Febbraio et al., 2002; 2004) and prolonged reduction of muscle

glycogen (Hesselink et al., 1998; Koh, 2002; 2004; Vissing et al., 2009) throughout recovery.

Skeletal muscle fibres contain large concentrations of cytoplasmic proteins. If the

sarcolemma or cell membrane becomes damaged, various cytoplasmic proteins and enzymes

escape and become detectable in the blood. The ease of collecting blood has allowed

investigators to use cytoplasmic proteins as markers of muscle damage (Schwane & Armstrong,

1983; King et al., 1993; Nosaka & Clarkson, 1996; Goldberg, 2003). However, blood markers of

muscle damage have been shown to be largely variable between and within subjects, making it

difficult to determine the amount of damaged muscle fibres (Nosaka & Clarkson, 1996; Kiang &

Tsokos, 1998; Sayers et al., 2003). Commonly reported proteins include: creatine kinase (CK),

myoglobin, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and tropoinin T.

2.2.4 Fiber-type differences

In both humans and animals, type II myofibres appear to suffer greater contraction-

induced damage than type I fibres (Fridén & Lieber, 2001; Gabai & Sherman, 2002). Type II

fibres may be more susceptible to damage due to a variety of reasons including: low oxidative

ATP turnover, lack of protective cytoskeletal structures and decreased protective heat shock


protein content (Skidmore et al., 1995; Patel et al., 1998; Koh, 2002; Tiidus, 2008). It is also

important to consider that structural fiber damage occurs specifically to contracting fibres

leaving non-contracting fibres mechanically intact (Newham et al., 1983; Cohen et al., 1991;

Fridén & Lieber, 1992; Enoka, 1996).

3.3.5 Symptoms of muscle damage

Contraction-induced muscle damage presents a variety of post-injury symptoms. The

most evident symptoms include: delayed onset muscle soreness (DOMS), edema, and reduced

range of motion (ROM). DOMS typically peaks 1-3 days post-injury and may be related to

biochemical products produced and released during the immune response (histamines,

prostaglandins and bradykinins) along with stretch-related hypersensitivity of nociceptors (Jones

et al., 1987; Smith, 1991; Febbraio et al., 2002; Cheung et al., 2003). Edema typically peaks 4-5

days following injury and gradually disseminates throughout the damaged tissue. Fluid

accumulation typically remains in the intracellular space between myofibres for an extended

period of time (5 days). Lastly, decreased ROM from damage is caused by shortening of the

muscle, although this has not been connected with increased neural activity (Nosaka & Clarkson,

1996; Thompson et al., 2001; 2003; Koh, 2004; Paulsen et al., 2007; 2009).

3.3.6 The Repeated Bout Effect

The repeated bout effect (RBE) is the term used to describe the rapid muscle fibre

protection gained from a single bout of eccentric contractions against muscle damage from

subsequent bouts of eccentric contractions (Nosaka & Clarkson, 1995; Tiidus, 2008). As

previously described, muscle fibre damage incurred from lengthening contractions results in a

period of impaired force-generating capabilities and also robust inflammatory response, which

often intensifies muscle tenderness. It has been demonstrated, in both humans and rats, that an


initial bout of eccentric contractions can protect against decreases in contractile force and

inflammation caused by subsequent eccentric contractions, lasting from a few weeks up until 6

months (Nosaka et al., 2001; Tiidus, 2008). Importantly, the protective adaptation is most

reliably induced following eccentric contractions of high intensity and not simply from a bout of

sub-maximal, high-volume eccentric contractions (Proske & Morgan, 2001; Nosaka & Newton,

2002; Tiidus, 2008). The mechanisms involved in this adaptation are still poorly understood, but

it is currently believed that protection is incurred though the enhancement of neural, mechanical

and biochemical systems (McHugh, 2003).

Since eccentric contractions typically require lower neural activity than concentric

contractions for a given muscle force, less motor units are required resulting in fewer contracted

fibres. This recruitment pattern results in more force exacted on a fewer number of contracted

muscle fibres. Neural adaptation following eccentric exercise has been suggested to involve an

altered pattern of motor unit recruitment to compensate for the imbalance in high forces applied

to a low number of fibres, which are highly susceptible to contraction-induced damage. Based

upon analysis in humans using electromyography (EMG), there is no change in signal amplitude

– indicating the theoretical recruitment of more motor units, but instead a decrease in signal

frequency possibly indicating neural synchronization or that low-force motor units are being

recruited (Clarkson & Tremblay, 1988; Faulkner et al., 1989; Warren et al., 1993; 2000;

McHugh et al., 2001; Tiidus, 2008).

It is also established that eccentrically contracted muscle rests with an increase in passive

and dynamic tension and decreased muscle length, described as mechanical adaptation.

Specifically, the cytoskeletal protein, desmin, which anchors and properly aligns sarcomeres, is

highly responsive to initial bouts of eccentric contractions (McHugh, 2003). Thus, investigations


on the physiological purpose of desmin disruption after lengthening muscle contractions and the

resultant adaptation has been studied. An increase in desmin content during the 3-7 days after

contraction-induced damage has been demonstrated (Barash et al., 2002). The investigators

speculated that increased desmin after damage would enhance adaptation by reinforcing

sarcomeres against increased loads experienced during subsequent contractions. This theory has

been the most widely accepted amongst researchers (Balnave & Allen, 1995; Warren et al.,

1999; Morgan & Allen, 1999; Allen, 2001; Warren et al., 2001; McHugh, 2003). Alternatively,

it was reported that mouse muscle lacking desmin through a gene knockout model was inflicted

with less myofibrillar disorganization after contraction compared to wild-type mice (Sam et al.,

2000). These results may indicate that certain cytoskeletal proteins, such as desmin, act as a

protective buffer and are thus important in decreasing sarcomere strain through their disruption

(Sam et al., 2000; Proske & Morgan, 2001). Lastly, it has been demonstrated that more

compliant, or forgiving muscle fibres experience less myofibrillar disruption, and that increased

desmin content results in less compliant fibres (McHugh et al., 1999). This ultimately means

that higher desmin content is generally associated with a less compliant muscle and thus

increased desmin content may not necessarily protect the muscle from damage. With regard to

the rapid adaptations of the RBE, disruption of desmin and other cytoskeletal proteins may

initially serve as a buffer to lessen inflicted damage, but later serve to reinforce sarcomeres after

content is increased.

Lastly, there are various cellular and biochemical adaptations believed to have an

important role in the RBE. Because contracted muscle length is an important factor in the

magnitude of inflicted muscle damage, the addition of sarcomeres in series serves to produce

more force and take strain at increased and more vulnerable muscle lengths (Morgan, 1990;


Warren et al., 1993; Lynn & Morgan, 1994; Lynn et al., 1998). Sarcomere addition has been

shown in humans and has also been proven to result in a rightward shift of the force-length curve

for sarcomeres, indicating higher force-generating capabilities at longer fibre lengths (Brockett et

al., 2001). As mentioned previously, the initial mechanical damage suffered from lengthening

contractions initiates a robust inflammatory response, which exacerbates the initial damage into a

more pronounced phase of recovery lasting up to 14 days depending on the intensity of

contraction. A blunted inflammatory response after contractions subsequent to initial damage

has been demonstrated, indicating the muscle has adapted and will not experience mechanical

damage to the same extent as a naïve muscle (Pizza et al., 1996). Nonetheless, a blunted

inflammatory response to contraction-induced damage results in less structural damage and

soreness – hallmark characteristics of the RBE.

Central to the present thesis, there are key cellular adaptive processes – namely the

induction of heat shock proteins (Hsps), which may also play a role in the repeated bout effect.

Hsps (discussed in section 3.4.3), serve an important function in protecting proteins from

denaturing during non-lethal cellular stress.

3.4 Heat Shock Proteins (Hsps)

Heat shock proteins (Hsps) are highly conserved molecular chaperones that are up-

regulated following various forms of external cellular stress (Welch, 1992). Initial reports

showed Hsp expression was increased after exposure to temperature elevations (Ritossa, 1962;

Tissières et al., 1974). It was then shown that Hsps are important for thermotolerance (Landry et

al., 1982) and protection against ischemia (Donnelly et al., 1992). Since these initial findings,

many other cellular stressors have been shown to increase the cellular content of Hsps including

ischemia (Marber et al., 1995), protein degradation (Chiang et al., 1989), hypoxia, acidosis


(Weitzel et al., 1985), oxidative stress (Davies et al., 1982; Chong et al., 1998), and energy

deprivation (Febbraio et al., 2002; 2004). Displayed in Table 1 are studies regarding Hsp

induction from a variety of cell stressors related to exercise. The common feature of most

stressors is hindered cellular function, which inherently subjects the cell to the deformation or

damage of functional and structural proteins. Many of the previously mentioned stressors are

present during a bout of exercise or throughout the subsequent recovery process.

Classification of Hsps is typically based on their molecular mass and range from small (8,

27kda) to large (90kda). There are numerous families of Hsps, which are expressed in muscle

and this section will describe their location, function and expression. The most commonly

studied include: Ubiquitin, αB-crystallin, Hsp25, Hsp60, Hsp72, Hsc70 and Hsp90, each of

which have previously been shown to have unique characteristics or roles and are commonly

associated with induction in specific cellular sub-compartments or organelles. Common Hsp

families along with their location and proposed function are compiled in Table 2, adopted from

Kregel (2002).

3.4.1 Hsp families and cellular functions

Due to their broadly protective nature and the previous work demonstrating an increase in

muscle concentrations following damaging contractions the most relevant heat shock proteins to

this thesis are Hsp25 and Hsp72.


Table 1 – Inducers of Hsps present during exercise

Type of Stressor Hsp Family Cell Type Model Reference

Acidosis Hsp70 Yeast cells in vitro Weitzel, 1985

Catecholamine Hsp70 Aorta tissue Rat (in vitro) Chin 1996

Increased Ca2+ Hsp80, Hsp100 Rat-1 cells Rat (in vitro) Welch, 1983

Ischemia/Hypoxia Hsp70 Cardiac muscle in vitro Mestril 1994

Metabolic Stress Hsp70 HeLa cells in vitro Beckmann, 1992

Hsp70 Skeletal muscle Human (in vivo) Febbraio, 2002

Hsp70 Skeletal muscle Human (in vivo) Febbraio, 2004

Hsp70 mRNA Skeletal muscle Human (in vivo) Febbraio & Koukoulas, 2000

Reactive Oxygen Species Hsp70 H9c2 myocytes in vitro Chong, 1998

Temp. Elevation Hsp70 Soleus muscle Rat (in vivo) Locke, 1990

Hsp70 Skeletal muscle Rat (in vivo) Skidmore, 1995

Hsp70, αB-crystallin Cardiac muscle Rat (in vivo) Harris & Starnes, 2001

Hsp70 Skeletal muscle Rat (in vivo) Paroo & Noble 1999


Table 2 – Hsp Families and their proposed functions

HSP family or Protein Cellular Locations Proposed Function

HSP27 (sHSP) Cytosol, nucleus Microfilament stabilization, antiapoptotic

HSP60 Mitochondria Refolds proteins and prevents aggregation of denatured proteins, proapoptotic

HSP70 Family: Antiapoptotic

HSP72 (Hsp70) Cytosol, nucleus Protein folding, cytoprotection

HSP73 (Hsc70) Cytosol, nucleus Molecular chaperones

HSP75 (mHSP70) Mitochondria Molecular Chaperones

HSP78 (GRP78) ER Cytoprotection, molecular chaperones

HSP90 Cytosol, ER, nucleus Regulation of steroid hormone receptors, protein translocation

HSP110/104 Cytosol Protein folding


Hsp25

Hsp25, or Hsp27 in humans, is named for its molecular mass of ~25kDa and is located in

small concentrations in the cytosol during unstressed conditions. Upon the application of

cellular stress, Hsp25 translocates into or around the nucleus (Arrigo et al., 1988). Initially, it

was determined that Hsp25 is involved in cellular functions such as molecular signal

transduction and cellular growth and differentiation (Arrigo & Landry, 1994). The induction of

Hsp25 originates from a single gene but post-translational phosphorylation allows for up to 4

isoelectric variants (Landry et al., 1989; 1992). Compared to other Hsps, Hsp25 accumulates at

a slower rate and is synthesized for a longer period after stress (Arrigo et al., 1988; Landry et al.,

1991). Specific to muscle fibres, Hsp25 has been shown to translocate and interact with

sections of the cytoskeleton and myofilaments after contraction-induced damage has been

inflicted (Koh, 2002; 2004; Vissing et al., 2009). In view of this, it has been speculated that

Hsp25 might stabilize the sarcomere and thereby have a protective role against any subsequent

damage. Furthermore, Hsp25 is believed to have a role in protecting cellular proteins from

oxidative stress (Escobedo et al., 2004). Oxidative stress is typically generated from prolonged

periods of increased metabolic demand such as with endurance-type exercise. A primary

component of oxidative stress is reactive oxygen species (ROS), which are known to have

denaturing effects on proteins (Goldberg, 2003). Thus, it is accepted that elevated muscle Hsp25

content occurs in response to denaturing and malfunctioning of proteins either from contraction-

induced damage or ROS.

Hsp70

Hsp70 refers to a family of Hsps including the constitutively expressed cognate protein,

Hsc73, and the highly stress-inducible 72kDa protein. Although both proteins share about a 90%


homology, Hsp72 appears to be of primary research interest due to its higher responsiveness and

resultant cellular abundance following stress. As the most highly conserved and extensively

studied family of Hsps, Hsp70 has demonstrated numerous cellular roles including the

preservation of cellular homeostasis (Gabai & Sherman, 2002). Although usually undetectable

in unstressed cells, Hsp70 is rapidly synthesized after stressors including: temperature increase

(Tissières et al., 1974) (Ritossa, 1962), acidity (Cohen et al., 1991), glycogen depletion

(Febbraio et al., 2002), and oxidative stress (Adrie et al., 2000). Hsp70 primarily serves as a

molecular chaperone providing cellular protection by maintaining protein structure and also by

preventing protein misfolding and aggregation (Kiang & Tsokos, 1998). In addition, isoforms of

Hsp70 serve as “molecular pivot” proteins, escorting newly synthesized, vulnerable proteins

between cell compartments or other molecular chaperones (Borges & Ramos, 2005). It has also

been demonstrated that Hsp70 interacts with the inhibitory IκB complex of NF-κB, a key

inflammatory transcription factor, that negatively regulates inflammatory cytokines (Malhotra &

Wong, 2002). Specific to this thesis, it has been repeatedly demonstrated that Hsp70 is

synthesized in skeletal muscle after exercise (Locke et al., 1990; Thompson et al., 2001; Khassaf

et al., 2001; Thompson et al., 2003; Koh, 2004; Morton, 2006; Paulsen et al., 2007; Morton et

al., 2009a; Paulsen et al., 2009). Interestingly, it has been shown that Hsp70 is highly expressed

in unstressed type I muscle fibres, while type II fibres, express low levels of Hsp70 in the

unstressed condition (Locke et al., 1991). Thus, it appears that the Hsp70 family provides

effective protein chaperoning and cellular protection, which may also aid during recovery after

contraction-induced damage.

eHsp70


Extracellular Hsp70 (eHsp70), defined by the presence of Hsp70 outside cells or in the systemic

circulation, has been shown to occur after heat stress and is linked with inflammatory activities.

Though not a major focus in this thesis, the concurrent expression of Hsp70 in skeletal muscle

and fibre membrane damage after lengthening muscle contractions make it likely that muscle

derived Hsp70 may diffuse outside of the muscle and function through Damage-associated

molecular patterns (DAMP) as eHsp70. Specifically, eHsp70 interacts with TLR2 and TLR4

membrane receptors on immune cells, which initiate the release of pro-inflammatory cytokines

such as TNF-α, IL-1β, IL-6 and IL-12 (Asea et al., 2002). Furthermore, eHsp70 can activate

NF-κB through the interaction with TLR4 by two separate signaling pathways: phosphorylation

of IκB and JNK/p38 (Asea et al., 2002).

3.4.2 Intracellular Molecular Functions of Hsps

Binding and regulatory processes of Hsps are graphically represented in Figure 2, adapted from

Noble (2008).

Intracellular Binding of Hsps

The classic theory of protein synthesis and denaturing of protein structure was initially

proposed by Anfinsen (1973). Synthesized proteins serve their intended function by folding in a

confirmation by which their active peptide groups can interact with their target substrate. During

synthesis, the primary amino acid structure of the protein folds into secondary “motifs” through

the interaction of individual amino acids within the protein before folding further into their

functional state. This amino acid interaction results in various bonds such as a “di-sulfide bond”

or hydrophobic/hydrophilic regions dictated by the collective polarity of the contained amino

acids. The complete structure of a properly functioning protein typically has hydrophobic amino

acid regions folded into the interior of the protein and hydrophilic regions on the exterior with


the functional or binding site readily accessible. The introduction of proteotoxic stressors such

as heat, ROS or acidity, denatures the complete protein and alters its structure often exposing

interior, hydrophobic regions to the exterior environment (Anfinsen, 1973). The unfolding of

proteins, or misfolding if the stressor is present during synthesis, will ultimately alter the

functional purpose of the protein and render it inactive. The aggregation of denatured proteins is

caused by the attraction of hydrophilic regions of one protein to the exposed hydrophobic regions

of other denatured proteins, which can cause cellular dysfunction and possibly apoptosis.

Heat shock proteins prevent protein denaturing and/or aggregation by their recognition

and high binding affinity to the properties of hydrophobic components of proteins. Once Hsps

have bound to hydrophobic regions, the host protein will remain functional even though the

bound Hsps have been introduced into the overarching protein structure (Borges & Ramos,

2005). In all cells, including skeletal muscle fibres, proper protein function is vital for proper

cell function making the activity of Hsps essential.

Regulation of the Cellular Stress Response

The regulation of cellular Hsp content is controlled by the location and DNA binding

ability of the responsible transcription factor, Heat shock factor 1 (HSF1). In unstressed cells,

HSF1 is transcriptionally inactive as a monomer bound to Hsp70 proteins in the cytosol

(Abravaya et al., 1992). Upon the detection of hydrophobic regions of denatured proteins, the

pool of free, unbound Hsps engages with the damaged proteins (Baler et al., 1992), leaving the

newly unbound HSF1 monomers free for trimerization and translocation into the nucleus where

it binds to short nucleotide sequences in DNA promoter sites of Hsp genes, called heat shock

elements (HSE) (Sarge et al., 1993). Once bound, HSF1 proceeds to up-regulate Hsp


expression, providing aid and protection to the cell. The ongoing production of new Hsps is

regulated through a negative feedback mechanism whereby newly translated Hsps start to appear

in excess due to Hsp content exceeding misfolded protein content (Morimoto, 1998). As such,

HSF1 nuclear activity is halted by DNA separation and re-binding to free Hsps in the cytosol.

3.4.3 Hsps and exercise

Endurance Exercise

Endurance exercise (EE) characteristically increases metabolic activity and oxygen

demand within working skeletal muscle. As such, energy deficits and metabolic byproducts are

produced hindering intracellular muscle function. Studies have demonstrated Hsp induction after

isolating or simulating various metabolic outcomes relating to EE, including: reduced glucose or

glycogen availability (Febbraio & Koukoulas, 2000; Febbraio et al., 2002), ATP depletion

(Beckmann et al., 1992), acidosis (Weitzel et al., 1985) and increased intracellular calcium

concentrations (Welch et al., 1983). Additionally, an increase in the cytosolic presence of

reactive oxygen species (ROS), generated and released from active mitochondria is speculated to

influence protein denaturation. However, the presence of ROS also directly influences the

activation of HSF1, thought to be the primary influence on synthesizing Hsps above that of

denatured proteins (Pattwell & Jackson, 2004).

Lastly, the increased body temperature experienced during prolonged endurance exercise

may not influence Hsp expression alone, as demonstrated in both rats (Skidmore et al., 1995) and

humans (Morton et al., 2007). Although somewhat surprising that increased body temperature


Figure 2: Intracellular HSP pathways - Adapted from original figure displayed in Noble et al.

(2008). Activation and intracellular heat shock response after exercise. 1. Mechanical

oxidative and metabolic stress or disturbances cause functional proteins to denature. 2. Heat

shock proteins (HSP70) bound to the Heat shock transcription factor (HSF1) release themselves

and 3. Bind to denatured proteins to aid in refolding. 4. Unbound HSF1 undergoes a series of

molecular events including trimerization, nuclear translocation and binding to the respective heat

shock elements (HSE). 5. HSPs are transcribed and become concentrated in cytosol, available

for the aided refolding of proteins. 6. Once denatured proteins have been addressed, a surplus of

free HSPs re-bind to HSF and deactivate the transcription factor. 1a. Alternative modes of HSP

transcription include intracellular signaling pathways such as Protein Kinase A (PKA) activation.

2a. PKA phosphorylates signaling proteins involved in sensing fuel storage.


from exercise does not seemingly influence Hsp expression, it is possible that a combination of

increased temperature with metabolic and oxidative stressors, as experienced during endurance

exercise, may together initiate the expression of Hsps.

Resistance exercise

Resistance exercise (RE) differs from EE because of an increased load and joint range of

motion (ROM). As discussed earlier, muscle subjected to overwhelming external loads and

unaccustomed fibre lengths are often damaged. Thus, performing RE significantly changes the

protein dynamics of the cell, manifested by the leakage of cytosolic proteins (i.e., creatine

kinase, myoglobin) along with membrane and sarcomere disruption and altered Ca2+

flux (Proske

& Morgan, 2001). Not surprisingly, the Hsp70 and Hsp25 families have been shown to be

upregulated during periods of increased muscle fibre overload in rodents (Oishi et al., 2005;

Ogata et al., 2005; O'Neill et al., 2006; Locke, 2008; Huey & Meador, 2008) and humans

(Thompson et al., 2001; 2003; Paulsen et al., 2007). Furthermore, following an initial bout of

fibre damage, Hsps have been shown to migrate to contractile protein structures, likely in an

effort to provide stabilization during recovery (Koh, 2004; Paulsen et al., 2007). Evidence of

damaged protein structures during overload suggests the importance of Hsps, which act in a

protective role and possibly enhance muscle adaptations to RE throughout recovery.

Hsps relating to the Repeated Bout Effect

Thomson et al. examined Hsps in relation to the RBE using resistance exercise performed

4 weeks apart. Hsp27 and Hsp70 content was evaluated, and increases above baseline were

observed after both bouts, although the baseline and absolute increase in both Hsps was


significantly lower compared to the initial bout (Thompson et al., 2002). The authors suspected

that the decreased fiber damage as indicated by decreased plasma CK concentrations after the

second bout influenced the diminished Hsp expression. It is possible that Hsp location following

the initial bout may also influence the diminished Hsp response. Paulsen et al. (2007) observed a

migration of Hsp27 towards the contractile myofibrillar structure following a bout of eccentric

contractions likely in an effort to protect against further damage. It is possible that the migration

and adherence of Hsps to contractile proteins from an initial bout may effectively lower the

stimulus for increased Hsp expression in subsequent bouts.

3.4.4 The influence of Hsps in muscle growth and loss

Hsps and Muscle Hypertrophy

Hsps may serve to enhance muscle hypertrophy over the long term given that rates of

muscle protein turnover (i.e., synthesis and breakdown) increase from exercise (Phillips et al.,

1997) and Hsps interaction with synthesizing proteins (Kiang & Tsokos, 1998). Using a chronic

overload model of muscle hypertrophy, Frier and Locke (2007) observed no further increase in

muscle growth in rats after the limb was heat stressed. No changes in muscle mass, protein

content and protein concentration were observed. It was speculated that the combination of heat

stress and the chronic overload model may have overstressed the “synthetic machinery” or

myogenic signaling proteins thereby limiting hypertrophy in the heat-stressed muscle.

Alternatively, concomitant increases in Hsp72 content and muscle hypertrophy have been

demonstrated using an occlusion-exercise model in rat skeletal muscle (Kawada & Ishii, 2005).

Hsp72 in the occluded limb muscle was significantly elevated above the muscles in the control

limb, which coincided with increased cross-sectional area (CSA) and myofibrillar protein


concentration. The exact role of Hsp72 during muscle hypertrophy was not entirely conclusive,

but it was demonstrated that both Hsp72 and muscle size can increase at the same time. Thus,

the exact role of Hsps during a positive net protein balance currently remains unclear.

Hsps Preventing Muscle Atrophy

Muscle atrophy occurs from chronic negative net protein balance, whereby protein

breakdown exceeds synthesis. Thus, limiting rates of protein breakdown is important when

aiming to minimize muscle atrophy and maximize growth. The importance of Hsp72 on muscle

preservation was examined by using prior heat shock followed by hind limb unloading in rats

(Naito et al., 2000). Heat shock induced a significant increase in Hsp72 over the control, which

resulted in significant preservation in muscle weight, myofibrillar protein and soluble protein

concentration above unloading alone. However, muscle preservation in the heat shock group

was still significantly lower compared with non heat-stressed controls that were not unloaded.

Moreover, a significant decrease in Hsp72 in the unloaded group suggests that increased Hsp72

expression may only respond to increased protein synthesis and not increased protein

breakdown.

3.4.5 Enhancing Athletic Performance

From an athletic performance standpoint, sustaining muscle force throughout competition

or training is the primary goal. Increased muscular temperatures (>40°C) commonly

experienced during exercise rapidly diminish contractile forces and results in fatigue.

Comparatively, decreased temperatures (<30°C) delay fatigue, allowing sustained contractile

forces (Bennett, 1984; Nguyen & Tidball, 2003). The fatiguing affect effect of increased muscle

temperature is observed through increased VO2, which may be caused by altered muscular O2


distribution, whereby the exterior muscle fibres starve interior fibres causing mild ischemia.

Although it would seem that prior heat shock, elevating Hsp70 and Hsp27 may attenuate protein

malfunction (Ca2+

, Na+/K

+ pumps) or degradation, unpublished data suggest that increased Hsp

content induced by heat stress does not conserve contractile force in abnormally high muscle

temperatures. In accordance with these findings, transgenic mice over-expressing Hsp72

experienced no protection against an intermittent fatiguing stimulation of extensor digitorum

longus (EDL) and soleus muscles (Nosek et al., 2000)

3.4.6 Cross-over effects of Hsp induction

The intracellular protein profile of skeletal muscle fibres includes a large portion of

myofibrillar-based protein and, to a lesser extent, metabolic-based protein in the form of

enzymes. Although Hsp induction is responsive to protein denaturation, different stressors elicit

different protective roles of Hsps. Furthermore, a general elevation in Hsps will not protect or

act against all types of stress. In a more direct examination of the cross-over effects of Hsp

induction, Thomas and Noble conducted a study showing that Hsps increased through heat shock

did not mediate any protective effect of force recovery following muscle fatigue (Thomas &

Noble, 1999). Thus, it remains to be fully determined if there is a cross-over effect of Hsp

protein protection between stressors that differ in nature.

4. Rationale

When challenged with protein-denaturing stressors such as heat stress or exercise,

skeletal muscle fibres synthesize heat shock proteins (Hsps) (Welch, 1992; Morton et al.,

2009b). While the exact functions of the various Hsp families remain to be determined, they are

believed to minimize protein denaturation and/or aggregation during periods of cellular stress


(Welch, 1992). Thus, much work has focused on identifying the roles and mechanisms of

induction of Hsps during or after exercise.

Lengthening contractions (LC, also known as eccentric contractions) are often performed

during daily activities and are associated with muscle fibre damage (Nosaka & Clarkson, 1995).

Damaged muscle exhibits decreased force (Brooks et al., 1995; Warren et al., 1999), plasma

membrane rupture (Fridén & Lieber, 1992; McNeil & Khakee, 1992), and myofibrillar

disorganization (Fridén et al., 1983; Fridén & Lieber, 1992). In response to the damage, the

muscle content of Hsp25 and Hsp72 appears to increase and translocate to the myofibrillar

structure, perhaps in an effort to aid or stabilize the sarcomere (Koh, 2004; Paulsen et al., 2007;

2009). In contrast to LCs, shortening contractions (SC, also called concentric contractions)

typically result in little if any fibre damage. However, it has been demonstrated that certain

outcomes or byproducts of continuous SCs such as elevated muscle temperature (Skidmore et

al., 1995), reactive oxygen species (ROS) (Davies et al., 1982; Salo et al., 1991; Chong et al.,

1998), and reduced glycogen availability (Febbraio et al., 2002) are indirectly capable of

inducing the cellular stress response.

Muscle Hsp content has been shown to increase in mammals following both LCs

(Thompson et al., 2001; 2003; Koh, 2004; Paulsen et al., 2007; 2009) and SCs (Khassaf et al.,

2001; Morton, 2006; Morton et al., 2009a); however, given the differences between the two

types of contractions, the accumulation patterns and underlying mechanisms of Hsp induction

between the contraction types may differ. With specific regards to SCs, perplexity exists in the

induction of Hsps as some investigators have not detected an increase in muscle Hsp content

after contractions (Febbraio & Koukoulas, 2000; Walsh et al., 2001; Febbraio et al., 2002). This

discrepancy is possibly explained by the use of treadmill exercise as the mode of contraction,


since it can vary in speed, duration, as well as the grade of incline or decline. In view of this, it

seems possible that LCs and SCs may initiate different patterns of muscle Hsp accumulation

when performed in a more controlled contraction setting. Thus, the purpose of the current study

was to define the temporal pattern of muscle Hsp25 and Hsp72 content up to 168h (7 days) after

a bout of 100 lengthening or shortening contractions, stimulated by 5 intermittent sets of 20

repetitions.

5. Objectives

1) To characterize temporal pattern of Hsp25 and Hsp72 protein content in the rat tibialis

muscle for 7-days after an electrically stimulated bout of 100 lengthening contractions

2) To compare the temporal pattern of Hsp25 and Hsp72 protein content after lengthening

contractions to a bout of 100 loaded shortening contractions, matched isokinetically to the

lengthening contractions.

6. Hypotheses

1) Hsp25 and Hsp72 content will be increased by 24h following the 100 lengthening and

shortening contractions.

2) The Hsp response will be greater in magnitude and duration after LCs due to muscle

damage. The HSP response from SCs will return to basal levels at 48h or 72h.

7. Methods & Materials

A diagram of the study design is presented in Figure 3

Animals.

Male Sprague-Dawley rats (N=65; 364.6±2.1g, mean±SEM) (Charles River Laboratories,

Wilmington, MA) were housed in pairs and maintained on a constant 12h light-dark cycle while

being fed and provided with water ad libitum. All procedures were approved by the Animal


Care Committee at the University of Toronto and were in accordance with Guidelines for

Canadian Council on Animal Care. The surgical and electrical stimulation procedures were

performed under anaesthesia (isoflurane/oxygen gas mixture; 1L/min). Following the electrically

stimulated contractions, animals were monitored for 1h while they recovered before being

returned to the animal care facilities until they were sacrificed by exsanguination under

anesthesia.

Study Design

Rats were grouped into either the shortening contractions (SC) treatment group (n=30), or the

lengthening contractions (LC) treatment group (n=30). The treatment groups were divided into

six subgroups representing progressive time points (2h, 8h, 24h, 48h, 72h, 168h) after the


Figure 3 – Schematic of Study Design


contraction bout to develop a temporal pattern of post-contraction muscle Hsp content. A

separate, non-stimulated control group of non-contracted rats (CON) (n=5) were also sacrificed

and their muscle used to represent basal Hsp conditions.

Stimulation protocol

Once anesthetized, animals were positioned in a supine position on a small-animal

warming platform (806D, Aurora Scientific Inc., Aurora, Canada) maintained at 37°C. Hair was

removed from the lower right hind limb with natural hair removal cream (Nair, USA). The right

knee was secured between two vertical stabilizing posts by delicately inserting a 25G x 1.5-inch

needle through the hind limb in a lateral orientation, directly distal to the condyles. Two 28G x

0.5-inch needle probe electrodes (Chalgren Enterprises. CA, USA) were inserted subcutaneously

in a longitudinal orientation, directly adjacent to the tibialis anterior (TA) muscle and the leg

was manually manipulated for ankle mobility and, more specifically, dorsiflexion. The right paw

was secured to the pedal of a computer-controlled servomotor (301C, Aurora Scientific Inc.,

Aurora, Canada) with adhesive tape. The pedal was adjusted three-dimensionally until the

secured ankle was in line with the knee and the knee angle was 120°. The pedal was then finely

adjusted to register zero-torque (g-cm) on the measurement software (DMC, Aurora Scientific

Inc., Aurora, Canada), signifying a neutral and squared leg position. The experimental setup is

illustrated in Figure 4.

Electrical stimulation was generated from a Grass Stimulator (S88, Grass Technologies.

RI, USA) controlled by a computer hardware interface (604A, Aurora Scientific. Aurora,

Canada) synchronized to the servomotor movements. Stimulation protocols and pedal

movements were arranged and executed with computer software (DMC, Aurora Scientific.


Figure 4: Graphical Representation of the Muscle Damage Model: Adapted from original

figure displayed in Tiidus (2008).


Aurora, Canada). Once the rat leg was set in the correct position, optimal stimulation voltage was

determined by stimulating the TA muscle to cause dorsiflexion of the paw against a rigid pedal

for 0.5-second periods in intervals of 1V, between 8 - 12V with 15 seconds rest between

stimulations. Isometric torque (g-cm) output was measured and the optimal stimulation voltage

for each rat was selected based upon the lowest voltage needed to elicit peak torque. Once peak

torque was achieved, the optimal stimulation frequency was determined in a similar manner by

keeping optimal voltage constant and increasing the frequency in intervals of 50Hz (100 -

300Hz) with rest periods of 15 seconds until maximal torque was optimized. Isometric torque

data from the single, optimized stimulation was collected and used as the pre-treatment peak

torque value. In most cases, optimal stimulation parameters were achieved before 5 muscle

contractions. Optimal stimulation parameters were typically between 8-10 volts and 150Hz.

Prior to each contraction during the treatment protocols, the servomotor passively

maneuvered the pedal along with the paw to a position where the ankle angle was 90o for LC or

120o for SC. The difference in starting limb positions for each contraction-type allowed for each

contraction to occur within the same range of motion. Electrical stimulation during the treatment

protocols was initiated for 0.2s prior to any servomotor movement to allow adequate muscular

force development and to collect isometric torque data for each individual contraction. While

remaining contracted, servomotor pedal movement was initiated either in the direction of plantar

flexion (LC) or dorsiflexion (SC) over a range of 38o

in 0.3 seconds, causing an angular

contraction velocity of 127o/s. With this experimental setup, the forced-lengthening of contracted

TA muscle in the LC group was isokinetically matched with the loaded shortening contractions

in the SC group. Upon the completion of each contraction, stimulation stopped and the pedal

passively maneuvered the paw back to the respective starting positions within 3 seconds. Both


LC and SC contraction protocols consisted of 100 stimulated contractions sectioned into 5 sets of

20, with each set separated by 5 min of rest. After the completion of the treatment protocols,

post-treatment peak torque was re-measured using a single 0.5-second stimulation against a rigid

pedal after 3 min and 10 min.

Tissue Collection

Rats were anaesthetized (isoflurane/oxygen gas mixture; 1L/min) and sacrificed by

exsanguination at various times (2, 8, 24, 48, 72, 168h) after SCs or LCs. The TA muscles from

both the contracted and non-contracted limbs were excised, weighed and divided into portions

for either histochemical or biochemical analyses. Muscle portions used for histochemical

analysis were oriented in a cross-sectional manner in OCT mounting gel and rapidly frozen with

isopentane previously cooled in liquid nitrogen. Portions used for Western blotting were quickly

frozen in liquid nitrogen. All samples were stored at -80oC until processed.

Fibre Morphology

Portions of frozen TA muscle in OCT gel were mounted in an American Optical cryostat

and cross-sections of 15-20μm were sliced, placed on microscope slides, air dried and stored at -

20°C. Slides were stained with Hematoxylin and Eosin (H&E) using standard techniques and

examined using a Zeiss Axioplot microscope at 40x magnification to identify muscle fibre

damage. Regions of fibre damage were identified by the presence of membrane rupture, swollen

cells – indicated by fibre rounding combined with noticeably darker-than-usual staining, “ghost

fibres” – indicated by a missing fibre surrounded by otherwise healthy-looking fibres and

mononuclear cell infiltration - indicated by swarming pattern of dark purple cells. Once

locations of fibre damage were identified, magnification was increased to 400X and images were


captured to represent the histopathology within these areas.

Protein Determination and Western Blot Analyses

Frozen portions of TA muscle (150-300 mg) were homogenized in 10 volumes of 600

mM NaCl and 15 mM Tris (pH 7.5) at 4°C using an Ultra-Turrax T8 grinder (IKA Labortechnik,

Staufen, Germany). Protein concentrations were determined by the method of Lowry et al.

(Lowry et al., 1951) using bovine serum albumin (BSA) as a standard. Based on the determined

protein concentrations, sample volumes containing 250μg of protein were loaded into 10%

acrylamide gels with one lane containing purified Hsp25 and Hsp72. Protein samples were

separated using one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE) according to the method described by Laemmli et al. (1970). Separated proteins

were transferred from the gel slab to nitrocellulose membranes (0.22 um pore size, Bio-Rad

Laboratories, Mississauga, Canada) using the method of Towbin et al. (1979), and modified to

the Bio-Rad mini-protean II gel transfer system as described by Frier et al. (2008).

Nitrocellulose membranes were blocked with 5% weight/volume non-fat skim milk power

(NFSM) dissolved in Tris-buffered saline (TBS) for 1 hour at room temperature. Blocked

membranes were washed twice for 5 min each with TBS plus Tween-20 (TTBS) before being

incubated overnight at 4°C with a polyclonal antibody specific for Hsp25 (ADI-SPP-715, ENZO,

USA) or Hsp72 (ADI-SPA-812, ENZO, USA) diluted 1:1000 in TTBS with 2% NFSM.

Incubated blots were washed twice in TTBS for 5 minutes. Membranes were incubated for 1

hour at room temperature with goat anti-rabbit secondary antibody conjugated to Horse-radish

peroxidase (HRP) (70745, Cell Signaling Technology, USA) in a 1:2500 dilution in TTBS and

2% NFSM. Membranes were washed twice in TTBS and once in TBS for 5 min each before

being treated with a luminol-based solution (Luminato Forte, Millipore, USA), exposed to film


(CLM5810, Bioflex, USA) and developed. Developed films were digitally scanned at 1200DPI

and band densities representing Hsp25 or Hsp72 content were quantified using ImageJ software

(version 1.43). Particular to Hsp25, both bands were quantified and counted as total Hsp25

content. Values obtained from scanned bands were compared to the contralateral, non-

contracted muscles and Hsp quantities were represented as fold-change from the contralateral

muscle. Hsp25 and Hsp72 in both TA muscle samples taken from the group of completely non-

contracted control rats was also quantified by western blotting and expressed as fold-change

from one another, representing basal Hsp content.

Statistics

A two-way ANOVA with independent measures was used to compare the interaction

effect of time vs. contraction type for Hsp25 and Hsp72. A two-way ANOVA with repeated

measures was used to compare the interaction effect of time vs. torque for all contraction torque

data, except inter-set decreases in torque, in which a paired t-test was used. A Bonferroni post-

hoc test was used to detect differences when the ANOVA revealed a significant interaction. All

data are reported as mean±SEM. The level of significance for all statistical tests was set at

P<0.05.

8. Results

Muscle mass

To assess whether the two contraction-types resulted in changes in muscle mass due to

swelling or edema, muscle mass was measured at the time of sacrifice (see Table 3). When SC

and non-stimulated muscle masses were compared, slight but significant increases (P<0.05) in

muscle masses of the SC group were detected at 2h (6.4%) and 8h (8.1%) but not thereafter.


After LCs, significant increases (P<0.05) in muscle mass in stimulated vs. non-stimulated

muscles were detected at 2h (6.4%), 8h (8.5%), 24h (7.8%), 48h (9.8%). At 72h, muscle mass

between LC muscles and contra-lateral muscles was similar but by 168h the mass of the LC

stimulated muscles was decreased (-9.7%) compared to contralateral controls. By expressing the

ratio of stimulated TA muscle mass by the total body mass (Table 3), comparisons between

contraction-types at each time point could be made. No significance at any time point (P>0.05)

was detected when comparing muscle mass after SCs to muscle mass after LCs. As a whole,

these data suggest that while both SCs and LCs showed initial elevations in muscle mass, the

mass of SC muscles returned to pre-contraction levels within a few hours while LCs resulted in a

sustained elevation of muscle mass that was followed by a drop in muscle mass.

Contractile Torque

To gain insight into how each contraction-type influenced muscle function, peak torque

(g-cm) was measured before the contraction bouts and at 3min and 10min after the 100

contractions (Table 4). The post-contraction peak torque data are also represented as a

percentage relative to the pre-contraction peak torque values for each animal (Figure 5). A

significant decrease in peak torque was detected at 3min (LC: 33.3±9.1%; SC: 63.2±11.7% of

pre-contraction values; P<0.05) and 10min (LC: 33.1±11.2%; SC: 68.4±12.9% of pre-

contraction values; P<0.05) after both contraction types. However, the decrease in peak torque

after the LCs was significantly greater when compared to SCs at both time points (P<0.05),

suggesting that there was an alternate mechanism causing contractile impairment with LCs such

as E-C impairment.


Table 3 – Muscle weight & Body weight

30

2h 5 364.4 ± 7.1 677.0 ± 9.7 * 636.0 ± 12.9 106.4 1.86 ± 0.06

8h 5 362.9 ± 4.5 659.6 ± 9.9 * 610.0 ± 35.2 108.1 1.82 ± 0.04

24h 5 368.7 ± 5.8 669.2 ± 20.0 664.2 ± 21.0 100.8 1.81 ± 0.04

48h 5 352.1 ± 9.0 633.2 ± 11.7 633.6 ± 15.7 99.9 1.80 ± 0.02

72h 5 363.8 ± 3.7 662.6 ± 15.5 665.6 ± 12.4 99.5 1.82 ± 0.05

168h 5 375.7 ± 8.9 663.6 ± 19.7 656.6 ± 15.2 101.1 1.77 ± 0.01

30

2h 5 362.5 ± 5.1 680.8 ± 29.4 * 640.0 ± 22.7 106.4 1.88 ± 0.06

8h 5 359.5 ± 3.8 683.0 ± 33.0 * 629.6 ± 28.5 108.5 1.90 ± 0.09

24h 5 349.2 ± 6.1 685.2 ± 19.2 * 635.8 ± 13.7 107.8 1.96 ± 0.03

48h 5 366.9 ± 6.0 691.0 ± 17.7 * 629.4 ± 12.8 109.8 1.88 ± 0.04

72h 5 370.3 ± 8.3 672.6 ± 22.1 661.6 ± 17.5 101.7 1.82 ± 0.04

168h 5 378.5 ± 10.6 596.2 ± 11.3 * 660.4 ± 26.2 90.3 1.58 ± 0.04

Shortening Contractions

Lengthening Contractions

Stimulated Muscle

mass/Body Mass (mg/g)

Percent mass

change from

control (%)

Non-Stimulated

TA muscle (mg)

Stimulated TA

muscle (mg)Body Mass (g)

n


Table 4 – Raw contraction data compiled

Pre-contractions

3min Post-contractions

10min Post-contractions

1st Rep 20th Rep 1st Rep 20th Rep

SET1 292.0 ± 13.5 202.2 ± 10.5 272.4 ± 8.0 171.3 ± 7.6

SET2 260.7 ± 11.6 205.6 ± 8.4 241.5 ± 5.5 115.0 ± 6.0

SET3 234.2 ± 12.8 183.0 ± 8.4 164.6 ± 6.9 78.0 ± 5.7

SET4 215.4 ± 13.1 167.1 ± 8.4 123.95 ± 7.7 57.4 ± 5.0

SET5 202.1 ± 11.6 156.6 ± 8.0 82.26 ± 5.8 42.5 ± 4.6

88.3 ± 5.6

89.2 ± 4.7

Shortening contractions Lengthening Contractions

193.0 ± 7.9

269.6 ± 6.57304.9 ± 6.36

Contraction Bout

209.0 ± 8.81


Figure 5 – Decreased Isometric Torque Following Both Shortening (SC) and Lengthening

Contractions (LC). Isometric torque measured at 3- and 10-minutes after the SC and LC bout

(expressed as values normalized to pre-contraction torque measurements). Values are means +/-

SEM; n=30 in each contraction type. #p<0.001 compared to pre-contraction torque; *p<0.001

compared to LC within time point.

3min 10min0

20

40

60

80

100

LC

SC

* *

#

Per

centa

ge

of

Pre

Pea

k T

orq

ue

(%)


Isometric torque (g-cm) measurements from the 1st and 20

th contraction of each completed set

are presented in Table 4. Additionally, isometric torque measured in the 20th

repetition of each

set was also normalized to the isometric torque measured in the 1st repetition of the 1

st set

(Figure 6 - panel A). A declining pattern of isometric torque from each set for both contraction-

types was observed. However, for each set the percentage decline in torque was significantly

(P<0.05) greater following LCs when compared to SCs. In addition, the 5 sets of LCs caused a

significantly (P<0.05) more rapid decline in isometric torque between each progressive set when

compared with SCs (LC: -11.7±0.78%; SC: -4.7±0.60%) (Figure 6 - panel B). Taken together,

these data indicate that LCs caused a more sustained impairment in contractile function than

SCs. This decreased occurred as early as the first set and continued throughout the entire

contraction bout.

Muscle Fiber Morphology

Hallmark characteristics of contraction-induced muscle fibre damage are illustrated in

Figure 7. Hematoxylin and Eosin staining was used to visualize TA muscle fibre morphology at

each time point after both contraction-types (Figure 8). Two hours after each contraction type no

changes in morphology were detected (Figure 8 - panels A and B) and the morphology was

similar to that observed for controls (data not shown). At 8h after LCs, muscle fibres showed

evidence of damage in the form of swollen fibres, vacant or “ghost fibres”, ruptured endomysium

as well as infiltration by mononuclear cells (Figure 8 - panel C). This pattern of fiber damage

after LCs continued up to 72h (Figure 8 - Panels C, E, G, I). In most cases, TA muscle fibres

were repaired and intact by 168h (Figure 8- panel K) after LCs and at this time point muscle

morphology was similar to the SC muscles and controls. However, some mononuclear cells were

still detected around regenerating muscle fibres at 168h (Figure 8 - panel K) post LC. In


Figure 6a – Decrease in Isometric Torque Throughout the Completion of Each Contraction-

type. Isometric torque measured during the 20th repetition of each set was normalized to isometric

torque measured during the initial contraction for each subject (1st rep of SET1). Values are means

+/- SEM; n=30 in each contraction type. *p<0.01 compared to LC within each SET

Figure 6b – Mean Decrease in Isometric Torque Between Consecutive Sets for Each

Contraction-type. Mean decrease in relative isometric torque between each set of contractions for

LC and SC. Values are represented as means ± SEM and expressed as percentage decline. *P<0.001

compared to SC.

SET1 SET2 SET3 SET4 SET5

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

*

**

*

*

LC

SCTorq

ue

Dec

reas

e fr

om

1st

RE

P (

%)

-15

-10

-5

0

LC

SC

*

Intr

a-se

t T

orq

ue

Dec

reas

e (%

)

A B


Figure 7 - Morphological Elements of Muscle Fibre Damage – Displayed images represent

different morphological elements of damage. A. Undamaged muscle fibres. B. Swollen muscle

fibre; darker staining, rounded appearance. C. Ghost fibres; absent fibre, accumulation of

infiltrating cells. D. Accumulation of infiltrating cells around various deformed fibres. The

displayed images were selected from the entire collection of samples sectioned and stained

during analysis.


Figure 8 – Stained cross-

sections representing fibre

morphology for the

contracted TA muscle.

Hemotoxylin and Eosin

stained tibialis anterior

cross-sections displaying

visual muscle fibre damage.

Visible fibre damage is

present at 8h after LC along

with a noticeable increase in

deep purple cells

disseminating around the

damaged fibres. These cells

are likely to be

inflammatory cells. No

visible fibre damage or

inflammatory cell

infiltration was observed

after SC.


contrast to the damage and disruption observed in muscle fibres following LCs, there was no

aberrant fiber morphology observed in muscles after SCs at any time point (Figure 8 – panels B,

D, F, H, J, L). These observations indicate that LCs resulted in muscle damage while SCs did

not result in any detectable muscle damage.

Muscle Hsp25 and Hsp72 protein content after muscle contractions

Hsp25 and Hsp72 were detected in all TA muscles examined by Western blot analyses.

Following quantification by densitometry, muscle Hsp content was expressed relative to the Hsp

content in the non-stimulated (contra-lateral) muscle of the same animal. A baseline or control

(CON) Hsp content was measured from the TA muscles of completely non-stimulated muscles

(controls). After the LCs, there was no significant differences detected in HSP25 content at 24h

(2.2±0.38-fold) or 72h (2.5±0.49-fold) when compared to controls, but significance was detected

at 48h (3.1±0.53 fold increase) and at 168h (3.0±0.83 fold increase) (Figure 9). When the

muscle Hsp25 content of the two contraction-types was compared within the specific time points,

significant (P<0.05) elevations in Hsp25 were detected between LCs and SCs at 24h and all

points thereafter. In contrast to the elevated Hsp25 observed after LCs, no significant increases in

Hsp25 content was observed at any time point following SCs.

Muscle Hsp72 content was also expressed relative to the contra-lateral control TA muscle

(Figure 10). When compared to CON, muscle Hsp72 content was significantly (P<0.05)

elevated at 24h, 48h and 72h by 3.8±0.66-, 2.6±0.49- and 3.22±0.57-fold, respectively for LC.

No increase in muscle Hsp72 content was observed after SCs for any of the time points

examined. When Hsp72 content between contraction-types within individual time points was

compared, a significant elevation (P<0.05) was detected at 8h (2.3±0.42 fold) and thereafter.


Taken together, these data suggest that electrically stimulated 100 LCs are capable of increasing

muscle Hsp content while 100 SCs does not result in an elevation of muscle Hsp25 or Hsp72

content.


Figure 9 – Intramuscular HSP25 Content After Lengthening or Shortening Contractions.

HSP25 protein expression measured 2h, 8h, 24h, 48h, 72h, 168h after the LC and SC bout (expressed

as fold change from values measured in contralateral control muscle). Values are means +/- SEM;

n=5 in each contraction type. *p<0.01 compared to SC within time point † p<0.05 compared to con

group. Representative blots are only contracted samples run along with purified HSP25.

con 2h 8h 24h 48h 72h 168h0

1

2

3

4

5

LC

SC

con 2h 8h 24h 48h 72h (3days)

168h (7days)

+con

*

*

*

*†

†

(3days) (7days)

HSP25

SC

LC

Fold

chan

ge

(fro

m c

ontr

ol

lim

b)


Figure 10 - Intramuscular HSP72 Content After Lengthening or Shortening Contractions.

HSP72 protein content measured 2h, 8h, 24h, 48h, 72h, 168h after the LC and SC bout (expressed as

fold change from values measured in contralateral control muscle). Values are means +/- SEM; n=5

in each contraction type. *p<0.05 compared to SC within time point † p<0.05 compared to con

group. Representative blots are only contracted samples run along with purified HSP72.

con 2h 8h 24h 48h 72h 168h0

1

2

3

4

5

con 2h 8h 1d 2d 3d 7d +con

EC

CC

*

*

*

*

*

†

†

†

con 2h 8h 24h 48h 72h(3days)

168h(7days)

+con

LC

SC

(3days) (7days)

HSP72

LC

SC

Fold

chan

ge

(fro

m c

ontr

ol

leg)


9. Discussion

The effect of 100 electrically stimulated shortening (non-damaging) or lengthening

(damaging) skeletal muscle contractions performed intermittently on the accumulation of

intramuscular Hsp25 and Hsp72 was investigated. The present experiment isolated the HSP

inducers specific to shortening and lengthening contractions from the multitude of inducers

generated during a typical bout of exercise. With the isolated model of muscle contraction, the

shortening contractions used in the present investigation could be isokinetically matched to the

lengthening contractions such that contraction velocity, time under tension and mechanical work

were all controlled. As hypothesized, forced lengthening contractions caused a significant

decrease in contractile function that was accompanied by an appreciable amount of muscle fibre

damage as well as a robust and sustained intramuscular Hsp response. Comparable shortening

contractions also resulted in significantly decreased contractile function, yet did not cause any

noticeable muscle fibre damage or elevations in Hsps. The highly controlled nature of the

contraction model allowed for the identification of the divergent patterns of Hsp25 and Hsp72

expression observed between lengthening and shortening contractions in the recovery period

from each contraction.

The degree of decreased contractile function can indicate the magnitude of muscle

damage (Warren et al., 1999). In the present study, we observed a significant decline (-38%) in

isometric torque in LC after the first set which was similar following SC (-31%). The patterns of

torque decline observed thereafter was different between contraction-types as demonstrated by a

greater decline in torque between each set (SC: -4.5% vs. LC: -11.7%; Figure 6b) and most

likely reflected excitation-contraction coupling impairment in the case of LC (Faulkner et al.,


1989; Warren et al., 1993; Brooks et al., 1995). The mild decrease in torque following SCs was

most likely caused by a mild progression of metabolic byproduct accumulation (Smith &

Newham, 2006) since no overt muscle damage was observed (Figure 8).

While LCs clearly resulted in muscle damage and an accumulation of Hsps, SCs showed

no change in Hsp accumulation. Due to the protective function of Hsps against stress, increases

in their expression can be linked with the magnitude of stress experienced by the cell.

Fundamentally, deformed and/or dysfunctional proteins characterize the initiation of the cell

stress response. When comparing the resultant fibre damage and hindered contractile function

after LCs to that observed after SCs, it is apparent that contraction-induced fibre damage inflicts

a significant level of perturbation from proteostasis whereas the SCs did not damage or denature

enough muscle proteins to elicit any Hsp accumulation. In the case of the lengthening

contractions reported herein, it is clear that accumulation of Hsp25 and Hsp72 is most likely

linked to the disruption of muscle fibres (and proteins) and possibly the infiltration and activity

of immune cells (Figure 8).

The differential vulnerability of fibre-types (i.e., type I or type II) to contraction-induced

damage has been addressed in earlier studies (Fridén & Lieber, 2001; Gabai & Sherman, 2002).

It has been proposed that type II fibres are more easily damaged by contraction based on their

low oxidative capacity (Fridén & Lieber, 1998) and capability to generate greater tensions

(Appell et al., 1992) compared to type I fibres . However, after increasing the oxidative capacity

of type II fibres through 4 weeks of electrical stimulation, it was found that type II fibres were

still susceptible to contraction-induced damage (Patel et al., 1998). It has also been proposed

that the innately shorter fibre length of type II muscle fibres results in greater fiber injury when

compared to type I fibres (Lieber & Fridén, 1999). It is known that type I muscle fibres contain


elevated levels of Hsp72, which likely contributes to their innate protection from contraction-

induced damage (Locke et al., 1991). In agreement with this, Tupling et al. (2007) reported that

compared to type II fibres, type I muscle fibres synthesize Hsps more rapidly (30 min vs. 1 day).

The TA muscle, as was used in the experiments reported herein, is known to consist of mostly

type II muscle fibres (95% Type II, 5% Type I) (Armstrong & Phelps, 1984). Choosing to

analyze the TA muscle, with a phenotype high in type II fibres allowed for significant damage

from the lengthening contractions and for the shortening contractions to elicit a sufficient level of

fatigue or metabolic stress. Since type II fibres also display low basal Hsp concentrations (Locke

et al., 1991), it was our expectation that any increases in Hsp content occurring specifically from

the intermittent shortening contractions would be detected.

In addition to resulting in a loss of contractile force, lengthening contractions are also

known to initiate robust inflammatory responses, whereby the muscle tissue is infiltrated by

neutrophils and macrophages (Nosaka & Clarkson, 1996; Frenette et al., 2002; McLoughlin et

al., 2003; Butterfield et al., 2006). This inflammatory response has been shown to exacerbate

initial contraction-induced fibre damage in the early (0-2 days) pro-inflammatory phase (Pizza et

al., 2001; 2004) but also aids with muscle recovery and adaptation later on (up to 14 days)

during the anti-inflammatory phase (Cantini et al., 1995; 2002; Tidball, 2004). The specific

function of immune cells (pro-inflammatory or anti-inflammatory) is guided by the presence and

activity of cytokines. For example, Tumor necrosis factor (TNF)-α is released from early-

responding neutrophils and acts by attracting more neutrophils and inducing the production of

other pro-inflammatory cytokines (Collins & Grounds, 2001; Zádor et al., 2001). There are

numerous molecular signals of inflammation and damage similar to TNF-α, termed “alarmins”,

which act as a sensor of tissue damage and also guide the inflammatory process (Bianchi, 2006).


The presence of Hsps in the extracellular environment have been proposed as important

alarmins, due to their interaction with several of the cellular receptors known to induce the

secretion of pro-inflammatory cytokines (Asea et al., 2000; Schmitt et al., 2006). The present

thesis demonstrates that neutrophils and macrophages infiltrated into damaged muscle fibres

between 8 - 168 hrs (Figure 8 - C, E, G, I, K) following LC, but no infiltration occurred at any

time point following SC. The temporal pattern of damaged fibres was accompanied by

elevations in Hsp25 (Figure 9) and Hsp72 content (Figure 10), which leads to the speculation

that the Hsps synthesized from within the fibre would diffuse passively into the extracellular

environment and influence the activity of the neutrophils and macrophages. Of course, since

Hsp25 and Hsp72 content was measured in total muscle homogenate, which includes immune

cells, it is possible that the Hsps originated from these cells along with damaged muscle fibres.

There is currently no evidence available, which would suggest that immune cells infiltrating

damaged muscle generate and contribute Hsps to the extracellular environment during recovery

from contraction-induced damage.

Although no increase in Hsp25 or Hsp72 was detected after the SCs in the present study,

other studies have reported the opposite effect (Khassaf et al., 2001; Morton, 2006; Morton et

al., 2009a). It was speculated that increased Hsps reported in these studies was associated

mainly with the generation and activity of ROS. The influence of ROS on muscle protein

oxidation and deformation was demonstrated by McArdle et al. (2001) who used a 15 minute

bout of isometric contractions in rats, which released superoxide ions (ROS) from muscle into

the extracellular space with a resultant transient oxidation of the thiol junctions of muscle

proteins. This study also demonstrated an increase in Hsp70 in the soleus and extensor

digitorum longus muscles. Furthermore, intracellular ROS are thought to be involved in


signaling transduction of the transcription factors: Nuclear Factor–κB (NF-κB) and Heat Shock

Factor 1 (HSF-1) (Pattwell & Jackson, 2004), which directly results in transcription of Hsps. To

further illustrate the influence of ROS on Hsp induction, antioxidants have been shown to

abolish or attenuate the level of Hsp70 induction from exercise (Khassaf et al., 2003; Jackson et

al., 2004). Although increased muscle temperature and depletion of intramuscular energy stores

are experienced during non-damaging contractions, there is evidence suggesting that these

factors do not influence Hsp induction in an exercise setting (Febbraio et al., 2002; Morton et al.,

2007). Lastly, it is also possible that the “stress response” is initiated after non-damaging

exercise as evident by elevated Hsp mRNA expression, but fails to elicit increases in actual Hsp

protein content (Puntschart et al., 1996; Walsh et al., 2001). Thus, Hsp expression may be

positively influenced by the intensity of non-damaging exercise, which was demonstrated by

Milne and Noble (2002) who reported that Hsp accumulation in rat white and red vastus

lateralus muscle is highly dependent on the speed of level treadmill running. Furthermore, the

effect of exercise intensity, involving non-damaging contractions, on Hsp accumulation was also

demonstrated in humans performing rowing exercise (Liu et al., 1999; 2000). Thus, it is

plausible that the shortening contractions performed intermittently herein were not of sufficient

intensity and, in turn, did not generate a metabolic stress substantial enough to influence the

induction of Hsps. It is likely that Hsp protein content will increase with non-damaging

contractions as long as a sufficient level or threshold of metabolic stress is reached as is more

likely during prolonged or high-intensity non-damaging exercise.

The muscular protection afforded by increased Hsps has been demonstrated using

transgenic mice overexpressing Hsp70 and other means. Maglara et al. (2003) demonstrated that

cultured muscle cells subjected to contraction-induced damage were protected by an elevated


Hsp70 content. In addition, another study using mice overexpressing Hsp70 demonstrated less

of a reduction in contractile force after contraction-induced damage in the extensor digitorum

longus muscle when compared to wild-type mice (McArdle et al., 2004). However, it is

important to note that the concentration of Hsp70 content in the skeletal muscle of

overexpressing mice were 20-fold above basal levels, likely enhancing any protective properties

compared with the ~3-fold increases observed after lengthening contractions in the current study.

More recently, an increased muscle Hsp content elicited by a prior heat shock, appeared to

enhance muscle reconditioning after fibre damage caused by downhill treadmill running

(Touchberry et al., 2012). The investigators reported lower plasma markers of muscle damage

along with increased total muscle protein concentration and myosin heavy chain protein content.

In response to fiber damage, both Hsp25 and Hsp72 appear to increase in concentration within

the muscle and translocate to the myofibrils after contraction-induced damage (Lavoie et al.,

1993; Koh, 2002; Paulsen et al., 2007; Vissing et al., 2009; Paulsen et al., 2009). Whether Hsps

are involved in attempting to stabilize the sarcomere remains to be determined but migration to

sarcomere structures does suggest that Hsps may aid in protection, repair and possibly muscle

hypertrophy. Indeed, an increased Hsp content from LCs or otherwise may benefit muscle

adaptation, and thus may have a therapeutic application for at-risk populations (i.e., frail,

dystrophic, etc.) (Lovering & Brooks, 2013). An elevated muscle Hsp content from LCs or

otherwise, may contribute to more rapid muscle adaptation, thus minimizing the effect of future

damaging muscle contractions - otherwise known as the repeated bout effect (RBE) (McHugh,

2003). At present, the only known method of minimizing the soreness, force decrement and

cellular damage from LCs is a prior, less severe bout of lengthening contractions/exercise. Hsp

have been proposed as a potential mechanism by which the RBE may minimize the damage


associated with LCs (McHugh, 2003). As demonstrated herein, non-damaging contractions do

not sufficiently elevate Hsps and thus will not experience any adaptive benefit or added

protection related to Hsp induction. Although the lengthening contractions were intended to

cause injury, it is also highly beneficial for exercise training regimens and therapeutic measures

to include a portion of lengthening contractions to enhance strength adaptation and maximize

muscle reconditioning (Lovering & Brooks, 2013).

10. Conclusion

In conclusion, controlled lengthening contractions of the rat TA muscle caused a marked

decrease in contractile function and caused visible muscle fiber damage while inducing a robust

and lasting increase in muscle Hsp25 and Hsp72 content. In contrast, isokinetically matched

shortening contractions caused a significant decline in contractile function, but did not elevate

Hsp25 or Hsp72 content. Thus, it appears that the stimulus for Hsp induction from intermittent

muscle contractions (i.e., resistance exercise) may occur from resultant fiber damage rather than

metabolically fatiguing, non-damaging muscle contractions. An increased Hsp content from

lengthening contractions may benefit muscle adaptation, and thus may have a therapeutic

application for at-risk populations (i.e., frail, dystrophic, etc.). At present, the only known

method of minimizing the soreness, force decrement and cellular damage from lengthening

contractions is a prior, less severe bout of lengthening contractions/exercise. Future directions

of research in the area of Hsp induction after contraction should include elucidating out the

influence of immune cells infiltrating into the damaged fibres. Additionally, the production or

appearance and influence of metabolic stressors such as ROS or depletion of energy stores using

the present model of intermittent muscle contractions should be more thoroughly investigated.

Lastly, there is currently a wide variability in the literature pertaining to the contraction model


used to study muscle adaptation after damaging and non-damaging contractions. A direct

comparison of stressors related to Hsp induction (heat, ROS, metabolic adaptation) between the

contraction model used herein and downhill or level treadmill running at various speeds is also

warranted.


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Appendix 1 – Laboratory Protocols

Analysis Protocol 1: Tissue Preparation and Sectioning Skeletal Muscle

Tissue Preparation

Materials:

1. 1 - Glass plate or flat surface – not the same ones for electrophoresis

2. 2 - sharp blades

3. 2 - 20 or 18G needles

4. Cork sections (~3cmx3cm) can use a wine cork and cut into sections

5. Dewar

6. Suspension apparatus

a. Sturdy stand with vertical bar

b. Horizontal arm secured to the vertical bar

c. Thin rope

7. Small metallic bowl (~250mL)

8. Tongs

9. Liquid Nitrogen (~1L)

10. Isopentane AKA methylbutane or 2-methylbutane (~150mL)

11. OCT compound (Optimal Cutting Temperature)

12. Tin foil

Preparation:

1. Label all cork pieces on the bottom for subject, condition, time, muscle

2. Set up thin rope and metal bowl on suspension apparatus above the doer of liquid

nitrogen

3. Pour isopentane (~150mL) into metal bowl and lower it into the liquid nitrogen to allow

the isopentane to cool (the bottom of the metal bowl will start to turn white)

Procedure:

1. Raise the metal bowl out of the liquid nitrogen.

2. Section muscle into 50-60mg pieces by using both blades in a scissor manner cutting

perpendicular to the fibres. Generally using the TA you can achieve 3 pieces.

3. Dab some OCT on the cork and submerse into the isopentane quickly to solidify the

outside layers, leaving the inside gelatinous.

4. Build up another layer of OCT by repeating the previous step.

5. Using the two needles, orient and submerge the muscle section with the fibres facing

directly upward.

6. Cover the muscle in OCT and submerge the complete preparation into isopentane (face

down)

7. Remove from isopentane and store in the liquid nitrogen while preparing the other

samples.

8. Remove from liquid nitrogen, wrap in tin foil and store in a separate container at -80°C


Sectioning

*Humidity and dull blades make sectioning extremely difficult

Materials

1. Cryostat, Myotome

2. Honeycomb block/chuck

3. Sharp blade (stored at -20°C)

4. Slides (can be stored at room temp or in the cryostat)

5. Paint brush (Chinese boar bristles, ¼ inch, flat or bright. cut at a 45° angle)

6. OCT compound

7. 95% Ethanol

8. Container for Ethanol

9. Slide Rack

10. Microscope

Preparation

1. Turn on the Cryostat with the chucks inside and close the access panel until -20°C is

reached

2. Take the blade (ensure it is sharpened) out of the freezer and position inside of the

cryostat. Tighten with the furthest knob on the left.

3. Remove the sample of interest from the cork and bond to the chuck with a dab of OCT

compound. To bond, leave the chuck inside of the cryostat for about 10-15min

4. Trim excess compound off of the periphery of the sample into a triangle shape with the

most narrow part to be cut first.

5. Insert the chuck into the holder and tighten.

Procedure

1. Adjust the angle of the blade to 4° with the black knob closest to you (most likely

unnecessary)

2. Adjusting the knob behind the curved ruler at the back of the cryostat will adjust section

thickness. (10-12μm is appropriate)

3. Adjust the blade cart height by locking and unlocking the lever closest to you.

4. Shave off a few sections to get nice, consistent slices and ensure the blade is cutting

properly.

5. Crank the black handle on the right hand side of the machine to cut slices. Ensure that

the cutting is even and not curling, folding or tearing (by crevasse in blade).

a. ENSURE that the axel does not become unhinged after too many cranks.

b. You can reset the axel by using the knob at the front of the machine and

readjusting the blade cart.

6. Using the paintbrush, grab the section as you slide 2mm above the bottom edge and pull

towards you to ensure a flat slice.

7. It is ideal to cut about 12 sections with the settings to keep consistency


8. Adhere to the slide by taking the room temperature slide and angling it at a 45deg angle

above the blade. As you slowly move the slide toward the section, it will adhere.

a. It is vital that drying time is minimized, as the tissue will undergo alterations

as it warms and dries.

9. As soon as the section has adhered to the slide, submerse in fixation solution (95%

ETOH) and then add to rack while cutting the rest of the sections

10. While cleaning the blade after slices, WIPE away so that you do not cut yourself.

11. Label and group slides in boxes.


Analysis Protocol 2: Hematoxylin and Eosin Staining

Materials:

1. Slide racks

2. Transfer containers and apparatus

Procedure:

Container # Reagent Time

1 Distilled Water (change every time) 1 min

2 Erlich’s Hematoxylin 7 min

3 Tap Water (change every time) 1 min

4 Distilled Water (change every time) 1 min

5 30% Ethanol 1 min

6 50% Ethanol 1 min

7 Eosin 30 sec

8 70% Ethanol (alternate the contents of this and the next dish

after each usage)

Rinse

9 70% Ethanol Rinse

10 80% Ethanol Rinse



13 100% Ethanol 2 min

14 100% Ethanol 5 min

15 100% Ethanol/Toluene 3 min

16 Toluene 5 min

17 Toluene 5 min

Notes:

- The line indicates where you can start the procedure

- Refresh the toluene in container #1 every 200 slides

- The Eosin and Hematoxylin should stain thousands of slides - It is advisable to filter the stains prior to use if they have been sitting for more than 2 days

(remove lumps)

- The Eosin may be refreshed with 70% ethanol and the Hemotoxylin with distilled water.


Analysis Protocol 3: Determination of Protein concentration – Lowry Assay

Lowry-Protein Determination

Standard H2Odd STD/Sample Reagent Phenol

or sample

0 5ml - - -

Blank 0.5ml - 5ml 0.5ml

20ug 0.48 20ul 5ml 0.5ml

40ug 0.46 40ul 5ml 0.5ml

60ug 0.44 60ul 5ml 0.5ml

80ug 0.42 80ul 5ml 0.5ml

100ug 0.40 100ul 5ml 0.5ml

SAMPLES: 0.500 5ul

1 0.500 5ul

2 0.500 5ul

3 0.500 5ul

4 0.500 5ul

etc 0.500 5ul

1ml (2%w/v) CuSO4.5H20.

48ml (3%w/v) Na2CO3 in 0.1 N NaOH.

16 tubes x 3 x 5 ml =240 ml

Therefore make up 250 ml reagent 5ml Na-T

5ml CuSO4.5H20

240 Na2CO3

1. In beaker, place 240 ml Na2CO3 , add Na-Tartrate first (5ml), then add CuSO4 (5ml).

2. Add standards and samples.

3. Add Lowry reagent, vortex , leave for a minimum of 10 min. (5ml/tube).

4. Mix Phenol reagent with dH20, 1:2. ie, 48 tubes need 24 ml (0.5 ml /tube)

-make up 27 ml ie, 9 ml phenol in 18 ml H2O dd.

5. Add 0.5 ml phenol reagent to each tube while mixing. Let sit for 0 min.

6. Read at 660 nm.


Analysis Protocol 4: SDS-PAGE and Western Blotting

1. Assemble pouring apparatus, Pour gels.

o 1mL APS => 0.1g APS and 1mL H2O

2. Load sample with 1:1 ratio of sample buffer

o Aim for 100μg/lane

o Run gel around 70-100V until dye front runs off the gel

3. Should take around 2.5h

o Ensure bubbles are present

o While gel is running gather the transfer materials (smaller casserole dish) and

make the

o Equilibrating buffer

4. Equilibrating buffer: 100mL 10x Running Buffer, 700mL ddH20, 200mL Methanol

o If not mixed in this order, salt will come out of solution

o Equilibrate gel and Nitrocellulose paper for 10min then set up transfer

sandwich

5. 2 notches in top of nitrocellulose: 1 diagonal for 1st well, 1 square for last well

6. Black, Brillo, 3 filters, GEL, Nitrocellulose, 3 filters, Brillo, Clear.

7. Ensure NO bubbles, use test tube to smooth

o Insert sandwiches and ice pack, fill with transfer buffer. Run at ~40V for 3h.

8. Ensure bubbles are present before turning on stir bar

9. Replace icepack ~1.5h into transfer

o Remove blot, block for 1-2hrs at room temp with % blotto

10. 10% Blotto: 10g blotto (skim milk powder), 100mL TBS

o 1st wash - TTBS (5min)

o 2nd wash - TTBS (5min)

o Incubate in 2% blotto with primary anti-body oscillating overnight in fridge

11. 1:1000 dilution of antibody:

o 2g blotto in 100mL TTBS, measure out 10mL

o Add 10μL antibody and vortex



o Incubate in 2% blotto with secondary anti-body for 1-2h at room temperature

12. 1:2500: 20mL blotto, 8μL antibody



o 3rd wash - TBS (5 min), drain wash, leave blot in container

o Treat Blot for either Alkaline Phosphatase or HRP visualization

o Add 5-6mL (for normal sized gel) of Luminata Forte HRP substrate incubate

for 5min

o Prepare the developing trays

13. Developer, Water, Fix

14. Can test the solutions by adding indicator to the fix, if cloudy make more

15. Dilution is on bottle (1 stock : 5 water, generally), make 1L

o Turn red light on, turn lights off, place card in exposure cassette, place film on


top

16. Expose for 1 min, 3 min, 5min, choose best one.

o Develop: 5min in Developer, 3min water, 3min Fix, 3min water, Dry.


Appendix 2 – Compiled Raw Data

Table 5 - Shortening Contractions


Table 6 - Lengthening Contractions

Time Subject Treatment Sacrifice Stim. Non-stim. Pre 3min 10min SET1 SET2 SET3 SET4 SET5 Stim. Non-Stim. Stim. Non-Stim.

2 h 1 350.0 350.0 599.0 553.0 248.8 58.6 60.3 101.0 100.4 37.5 27.7 14.8 44603.6 47347.6 1359.5 939.4

2 h 2 363.5 363.5 748.0 661.0 266.7 82.8 85.5 109.1 141.1 86.2 62.6 47.5 1825.5 1728.5 9875.6 10496.3

2 h 3 373.0 373.0 745.0 685.0 286.4 113.5 119.1 75.3 114.6 97.9 66.2 40.3 61014.3 57315.4 20567.1 16028.5

2 h 4 374.5 374.5 675.0 655.0 298.1 159.1 153.8 120.3 93.3 99.0 72.8 66.8 48459.0 41465.2 12840.7 15922.9

2 h 5 351.5 351.6 637.0 646.0 266.6 134.5 131.5 74.8 85.8 77.8 35.4 19.2 40978.8 33758.6 1824.7 1603.5

8 h 1 352.0 352.0 655.0 614.0 274.1 102.2 114.6 111.6 126.7 104.8 82.7 54.9 38230.9 37803.4 13909.5 6609.2

8 h 2 363.0 363.0 810.0 741.0 226.6 98.8 110.2 105.4 151.0 118.2 82.5 64.3 7019.9 5274.0 28060.0 15903.0

8 h 3 371.5 371.5 621.0 595.0 227.6 42.4 29.2 105.1 139.6 60.1 42.4 22.2 46208.4 29316.9 39545.5 29314.4

8 h 4 360.0 360.0 651.0 616.0 315.2 59.4 48.5 113.6 134.5 49.9 10.6 -15.9 43648.3 37467.8 38554.5 10143.3

8 h 5 351.0 351.0 678.0 582.0 325.2 72.3 50.0 103.0 149.3 78.2 36.8 3.0 29640.6 19584.1 13962.0 5506.0

24 h 1 351.0 354.0 686.0 595.0 267.2 72.8 77.3 95.0 136.0 107.4 70.2 56.6 36136.0 13793.0 29986.4 6215.3

24 h 2 338.9 345.9 659.0 615.0 209.2 63.5 82.5 147.9 134.9 93.9 80.9 53.1 41058.9 13542.9 33582.4 18241.4

24 h 3 369.3 363.2 757.0 672.0 313.0 130.4 137.8 113.9 128.8 93.5 81.7 71.1 72628.1 50901.6 43440.3 8430.6

24 h 4 327.0 327.5 647.0 651.0 252.7 107.2 111.8 81.2 106.8 82.9 76.3 50.2 87408.5 31003.4 50708.5 10908.5

24 h 5 356.0 355.5 677.0 646.0 300.1 96.7 90.6 113.6 114.3 73.7 38.6 21.6 71185.6 60836.9 45327.6 16820.7

48 h 1 367.9 377.6 704.0 630.0 279.7 62.0 53.3 101.7 137.7 98.0 64.3 38.3 52865.4 18209.7 8184.9 4223.5

48 h 2 360.0 369.5 738.0 601.0 339.6 98.4 100.4 129.5 143.4 77.3 51.1 28.0 26462.6 7152.8 42306.4 15344.8

48 h 3 356.3 370.0 644.0 616.0 261.2 113.5 119.1 96.7 115.7 112.1 95.5 76.1 90082.2 20146.4 8274.3 3948.1

48 h 4 374.4 343.5 656.0 623.0 316.3 80.0 77.3 142.4 166.7 93.1 79.2 41.7 88841.4 28135.4 42866.6 9600.2

48 h 5 361.0 374.0 713.0 677.0 274.7 82.4 78.8 72.6 138.9 83.2 N/A 45.7 74941.5 59272.6 45927.5 24653.1

72 h 1 332.5 342.5 616.0 609.0 244.5 69.1 74.3 91.8 102.2 87.6 61.1 36.7 42164.9 26526.7 34973.3 10037.3

72 h 2 345.5 363.2 649.0 655.0 238.9 77.9 73.6 85.4 122.0 86.3 71.7 55.8 31188.5 8872.9 18079.1 16474.4

72 h 3 372.5 385.0 739.0 695.0 306.5 117.8 124.5 106.8 135.4 101.3 61.2 39.2 79713.9 22348.3 47732.6 12777.7

72 h 4 351.5 372.5 707.0 705.0 227.1 89.8 93.5 81.7 128.7 96.7 70.9 57.8 79525.9 32213.3 46074.1 10324.9

72 h 5 362.5 388.5 652.0 644.0 265.3 77.6 55.2 103.5 139.0 74.4 51.0 33.5 62890.1 53544.2 58745.5 17552.2

168 h 1 310.0 372.5 589.0 610.0 237.8 100.7 99.1 80.3 109.1 100.8 71.9 40.1 56164.7 9581.8 36040.4 10202.9

168 h 2 332.0 391.0 637.0 674.0 238.9 75.9 71.2 123.4 141.9 68.3 68.9 47.1 32669.3 8443.0 19944.9 14694.7

168 h 3 353.6 405.0 601.0 742.0 268.0 102.5 111.6 88.2 138.3 105.8 69.3 43.3 82885.6 43802.3 49766.7 13401.9

168 h 4 309.0 342.0 584.0 597.0 200.5 72.8 65.0 37.1 99.6 80.9 49.5 24.9 34430.4 18396.0 41452.2 40777.8

168 h 5 322.0 382.0 570.0 679.0 310.7 62.8 48.8 121.5 121.4 70.4 34.0 14.8 70790.8 50211.4 60126.1 34428.4

HSP72 (arb. Units)Body Mass (g) TA mass (mg) Torque Tests (g-cm) Δ Torque 1rep-20rep (g-cm) HSP25 (arb.units)

A COMPARISON OF HEAT SHOCK PROTEIN EXPRESSION IN RAT ... · evaluating muscle Hsp expression in the...

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