Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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
Holwerda, A - M.Sc Thesis 2
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
Holwerda, A - M.Sc Thesis 3
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
Holwerda, A - M.Sc Thesis 4
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).
Holwerda, A - M.Sc Thesis 5
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
Holwerda, A - M.Sc Thesis 6
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).
Holwerda, A - M.Sc Thesis 7
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
Holwerda, A - M.Sc Thesis 8
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
Holwerda, A - M.Sc Thesis 9
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
Holwerda, A - M.Sc Thesis 10
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
Holwerda, A - M.Sc Thesis 11
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;
Holwerda, A - M.Sc Thesis 12
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
Holwerda, A - M.Sc Thesis 13
(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.
Holwerda, A - M.Sc Thesis 14
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
Holwerda, A - M.Sc Thesis 15
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
Holwerda, A - M.Sc Thesis 16
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%
Holwerda, A - M.Sc Thesis 17
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
Holwerda, A - M.Sc Thesis 18
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
Holwerda, A - M.Sc Thesis 19
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
Holwerda, A - M.Sc Thesis 20
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
Holwerda, A - M.Sc Thesis 21
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.
Holwerda, A - M.Sc Thesis 22
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
Holwerda, A - M.Sc Thesis 23
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
Holwerda, A - M.Sc Thesis 24
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
Holwerda, A - M.Sc Thesis 25
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
Holwerda, A - M.Sc Thesis 26
(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,
Holwerda, A - M.Sc Thesis 27
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
Holwerda, A - M.Sc Thesis 28
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
Holwerda, A - M.Sc Thesis 29
Figure 3 – Schematic of Study Design
Holwerda, A - M.Sc Thesis 30
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.
Holwerda, A - M.Sc Thesis 31
Figure 4: Graphical Representation of the Muscle Damage Model: Adapted from original
figure displayed in Tiidus (2008).
Holwerda, A - M.Sc Thesis 32
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
Holwerda, A - M.Sc Thesis 33
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
Holwerda, A - M.Sc Thesis 34
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
Holwerda, A - M.Sc Thesis 35
(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.
Holwerda, A - M.Sc Thesis 36
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.
Holwerda, A - M.Sc Thesis 37
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
Holwerda, A - M.Sc Thesis 38
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
Holwerda, A - M.Sc Thesis 39
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
(%)
Holwerda, A - M.Sc Thesis 40
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
Holwerda, A - M.Sc Thesis 41
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
Holwerda, A - M.Sc Thesis 42
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.
Holwerda, A - M.Sc Thesis 43
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.
Holwerda, A - M.Sc Thesis 44
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.
Holwerda, A - M.Sc Thesis 45
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.
Holwerda, A - M.Sc Thesis 46
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)
Holwerda, A - M.Sc Thesis 47
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)
Holwerda, A - M.Sc Thesis 48
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.,
Holwerda, A - M.Sc Thesis 49
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
Holwerda, A - M.Sc Thesis 50
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).
Holwerda, A - M.Sc Thesis 51
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
Holwerda, A - M.Sc Thesis 52
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
Holwerda, A - M.Sc Thesis 53
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
Holwerda, A - M.Sc Thesis 54
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
Holwerda, A - M.Sc Thesis 55
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.
Holwerda, A - M.Sc Thesis 56
11. References
Abravaya K, Myers MP, Murphy SP & Morimoto RI (1992). The human heat shock protein
hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression.
Genes Dev 6, 1153–1164.
Adrie C, Richter C, Bachelet M, Banzet N, François D, Dinh-Xuan AT, Dhainaut JF, Polla BS &
Richard MJ (2000). Contrasting effects of NO and peroxynitrites on HSP70 expression and
apoptosis in human monocytes. Am J Physiol, Cell Physiol 279, C452–C460.
Allen DG (2001). Eccentric muscle damage: mechanisms of early reduction of force. Acta
Physiol Scand 171, 311–319.
Anfinsen CB (1973). Principles that govern the folding of protein chains. Science 181, 223–230.
Appell HJ, Soares JM & Duarte JA (1992). Exercise, muscle damage and fatigue. Sports Med 13,
108–115.
Armstrong RB & Phelps RO (1984). Muscle fiber type composition of the rat hindlimb. Am J
Anat 171, 259–272.
Arrigo A-P & Landry J (1994). Expression and Function of the Low-molecular-weight Heat
Shock Proteins. Cold Spring Harbor Monograph Archive 26, 335–373.
Arrigo AP, Suhan JP & Welch WJ (1988). Dynamic changes in the structure and intracellular
locale of the mammalian low-molecular-weight heat shock protein. Mol Cell Biol 8, 5059–
5071.
Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC &
Calderwood SK (2000). HSP70 stimulates cytokine production through a CD14-dependant
pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 6, 435–442.
Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA & Calderwood SK
(2002). Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like
receptor (TLR) 2 and TLR4. J Biol Chem 277, 15028–15034.
Atomi Y, Yamada S, Strohman R & Nonomura Y (1991). Alpha B-crystallin in skeletal muscle:
purification and localization. Journal of Biochemistry 110, 812–822.
Baler R, Welch WJ & Voellmy R (1992). Heat shock gene regulation by nascent polypeptides
and denatured proteins: hsp70 as a potential autoregulatory factor. The Journal of Cell
Biology 117, 1151–1159.
Balnave C & Allen D (1995). Intracellular calcium and force in single mouse muscle fibres
following repeated contractions with stretch. The Journal of Physiology 488, 25–36.
Balnave CD, Davey DF & Allen DG (1997). Distribution of sarcomere length and intracellular
calcium in mouse skeletal muscle following stretch-induced injury. The Journal of
Holwerda, A - M.Sc Thesis 57
Physiology 502 ( Pt 3), 649–659.
Barash IA, Peters D, Fridén J, Lutz GJ & Lieber RL (2002). Desmin cytoskeletal modifications
after a bout of eccentric exercise in the rat. Am J Physiol Regul Integr Comp Physiol 283,
R958–R963.
Beckmann RP, Lovett M & Welch WJ (1992). Examining the function and regulation of hsp 70
in cells subjected to metabolic stress. The Journal of Cell Biology 117, 1137–1150.
Bennett A (1984). Thermal dependence of muscle function. Am J Physiol Regul Integr Comp
Physiol 247, R217–R229.
Bianchi ME (2006). DAMPs, PAMPs and alarmins: all we need to know about danger. Journal
of Leukocyte Biology 81, 1–5.
Borges JC & Ramos CHI (2005). Protein folding assisted by chaperones. Protein Pept Lett 12,
257–261.
Brockett CL, Morgan DL & Proske U (2001). Human hamstring muscles adapt to eccentric
exercise by changing optimum length. Medicine & Science in Sports & Exercise 33, 783–
790.
Brooks SV, Zerba E & Faulkner JA (1995). Injury to muscle fibres after single stretches of
passive and maximally stimulated muscles in mice. The Journal of Physiology 488 ( Pt 2),
459–469.
Brown LM & Hill L (1991). Some observations on variations in filament overlap in tetanized
muscle fibres and fibres stretched during a tetanus, detected in the electron microscope after
rapid fixation. J Muscle Res Cell Motil 12, 171–182.
Butterfield TA, Best TM & Merrick MA (2006). The dual roles of neutrophils and macrophages
in inflammation: a critical balance between tissue damage and repair. J Athl Train 41, 457–
465.
Cantini M, Giurisato E, Radu C, Tiozzo S, Pampinella F, Senigaglia D, Zaniolo G, Mazzoleni F
& Vitiello L (2002). Macrophage-secreted myogenic factors: a promising tool for greatly
enhancing the proliferative capacity of myoblasts in vitro and in vivo. Neurol Sci 23, 189–
194.
Cantini M, Massimino ML, Rapizzi E, Rossini K, Catani C, Dalla Libera L & Carraro U (1995).
Human satellite cell proliferation in vitro is regulated by autocrine secretion of IL-6
stimulated by a soluble factor(s) released by activated monocytes. Biochem Biophys Res
Commun 216, 49–53.
Cheung K, Hume P & Maxwell L (2003). Delayed onset muscle soreness : treatment strategies
and performance factors. Sports Med 33, 145–164.
Chiang HL, Terlecky SR, Plant CP & Dice JF (1989). A role for a 70-kilodalton heat shock
Holwerda, A - M.Sc Thesis 58
protein in lysosomal degradation of intracellular proteins. Science 246, 382–385.
Chong KY, Lai CC, Lille S, Chang C & Su CY (1998). Stable overexpression of the constitutive
form of heat shock protein 70 confers oxidative protection. J Mol Cell Cardiol 30, 599–608.
Clarkson PM & Tremblay I (1988). Exercise-induced muscle damage, repair, and adaptation in
humans. J Appl Physiol 65, 1–6.
Cohen DS, Palmer E, Welch WJ & Sheppard D (1991). The response of guinea pig airway
epithelial cells and alveolar macrophages to environmental stress. Am J Respir Cell Mol Biol
5, 133–143.
Collins RA & Grounds MD (2001). The Role of Tumor Necrosis Factor-alpha (TNF-α) in
Skeletal Muscle Regeneration Studies in TNF-α (-/-) and TNF-α (-/-)/LT-α (-/-) Mice.
Journal of Histochemistry & Cytochemistry 49, 989–1001.
Davies KJ, Quintanilha AT, Brooks GA & Packer L (1982). Free radicals and tissue damage
produced by exercise. Biochem Biophys Res Commun 107, 1198–1205.
Donnelly TJ, Sievers RE, Vissern FL, Welch WJ & Wolfe CL (1992). Heat shock protein
induction in rat hearts. A role for improved myocardial salvage after ischemia and
reperfusion? Circulation 85, 769–778.
Enoka RM (1996). Eccentric contractions require unique activation strategies by the nervous
system. J Appl Physiol 81, 2339–2346.
Escobedo J, Pucci AM & Koh TJ (2004). HSP25 protects skeletal muscle cells against oxidative
stress. Free Radical Biology and Medicine 37, 1455–1462.
Faulkner JA, Jones DA & Round JM (1989). Injury to skeletal muscles of mice by forced
lengthening during contractions. Q J Exp Physiol 74, 661–670.
Febbraio M & Koukoulas I (2000). HSP72 gene expression progressively increases in human
skeletal muscle during prolonged, exhaustive exercise. J Appl Physiol 89, 1055–1060.
Febbraio MA, Mesa JL, Chung J, Steensberg A, Keller C, Nielsen HB, Krustrup P, Ott P, Secher
NH & Pedersen BK (2004). Glucose ingestion attenuates the exercise-induced increase in
circulating heat shock protein 72 and heat shock protein 60 in humans. Cell Stress and
Chaperones 9, 390–396.
Febbraio MA, Steensberg A, Walsh R, Koukoulas I, van Hall G, Saltin B & Pedersen BK (2002).
Reduced glycogen availability is associated with an elevation in HSP72 in contracting
human skeletal muscle. The Journal of Physiology 538, 911–917.
Fielding RA, Manfredi TJ, Ding W, Fiatarone MA, Evans WJ & Cannon JG (1993). Acute phase
response in exercise. III. Neutrophil and IL-1 beta accumulation in skeletal muscle.
Frenette J, St-Pierre M, Côté CH, Mylona E & Pizza FX (2002). Muscle impairment occurs
Holwerda, A - M.Sc Thesis 59
rapidly and precedes inflammatory cell accumulation after mechanical loading. Am J Physiol
Regul Integr Comp Physiol 282, R351–R357.
Fridén J & Lieber RL (1992). Structural and mechanical basis of exercise-induced muscle injury.
Medicine & Science in Sports & Exercise 24, 521–530.
Fridén J & Lieber RL (1998). Segmental muscle fiber lesions after repetitive eccentric
contractions. Cell and Tissue Research 293, 165–171.
Fridén J & Lieber RL (2001). Eccentric exercise-induced injuries to contractile and cytoskeletal
muscle fibre components. Acta Physiol Scand 171, 321–326.
Fridén J, Sjöström M & Ekblom B (1983). Myofibrillar damage following intense eccentric
exercise in man. Int J Sports Med 4, 170–176.
Frier BC & Locke M (2007). Heat stress inhibits skeletal muscle hypertrophy. Cell Stress and
Chaperones 12, 132–141.
Frier BC, Noble EG & Locke M (2008). Diabetes-induced atrophy is associated with a muscle-
specific alteration in NF-κB activation and expression. Cell Stress and Chaperones 13, 287–
296.
Gabai VL & Sherman MY (2002). Invited review: Interplay between molecular chaperones and
signaling pathways in survival of heat shock. J Appl Physiol 92, 1743–1748.
Goldberg AL (2003). Protein degradation and protection against misfolded or damaged proteins.
Nature 426, 895–899.
Hesselink M, Kuipers H & Keizer H (1998). Acute and sustained effects of isometric and
lengthening muscle contractions on high-energy phosphates and glycogen metabolism in rat
tibialis anterior muscle. J Muscle Res Cell Motil 19, 373–380.
Huey KA & Meador BM (2008). Contribution of IL-6 to the Hsp72, Hsp25, and -crystallin
responses to inflammation and exercise training in mouse skeletal and cardiac muscle.
Journal of Applied Physiology 105, 1830–1836.
Ingalls CP, Warren GL & Armstrong RB (1998). Dissociation of force production from MHC
and actin contents in muscles injured by eccentric contractions. J Muscle Res Cell Motil 19,
215–224.
Jackson MJ, Khassaf M, Vasilaki A, McArdle F & McArdle A (2004). Vitamin E and the
Oxidative Stress of Exercise. Annals of the New York Academy of Sciences 1031, 158–168.
Jones DA, Newham DJ & Clarkson PM (1987). Skeletal muscle stiffness and pain following
eccentric exercise of the elbow flexors. Pain 30, 233–242.
Kawada S & Ishii N (2005). Skeletal Muscle Hypertrophy after Chronic Restriction of Venous
Blood Flow in Rats. Medicine & Science in Sports & Exercise 37, 1144–1150.
Holwerda, A - M.Sc Thesis 60
Khassaf M, Child RB, McArdle A, Brodie DA, Esanu C & Jackson MJ (2001). Time course of
responses of human skeletal muscle to oxidative stress induced by nondamaging exercise. J
Appl Physiol 90, 1031–1035.
Khassaf M, McArdle A, Esanu C, Vasilaki A, McArdle F, Griffiths RD, Brodie DA & Jackson
MJ (2003). Effect of vitamin C supplements on antioxidant defence and stress proteins in
human lymphocytes and skeletal muscle. The Journal of Physiology 549, 645–652.
Kiang JG & Tsokos GC (1998). Heat shock protein 70 kDa: molecular biology, biochemistry,
and physiology. Pharmacol Ther 80, 183–201.
King DS, Feltmeyer TL, Baldus PJ, Sharp RL & Nespor J (1993). Effects of eccentric exercise
on insulin secretion and action in humans. J Appl Physiol 75, 2151–2156.
Koh TJ (2002). Do small heat shock proteins protect skeletal muscle from injury? Exerc Sport
Sci Rev 30, 117.
Koh TJ (2004). Cytoskeletal disruption and small heat shock protein translocation immediately
after lengthening contractions. AJP: Cell Physiology 286, 713C–722.
Kregel KC (2002). Invited review: heat shock proteins: modifying factors in physiological stress
responses and acquired thermotolerance. J Appl Physiol 92, 2177–2186.
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680–685.
Landry J, Bernier D, Chrétien P, Nicole LM, Tanguay RM & Marceau N (1982). Synthesis and
degradation of heat shock proteins during development and decay of thermotolerance.
Cancer Res 42, 2457–2461.
Landry J, Chrétien P, Lambert H, Hickey E & Weber LA (1989). Heat shock resistance
conferred by expression of the human HSP27 gene in rodent cells. The Journal of cell ….
Landry J, Chrétien P, Laszlo A & Lambert H (1991). Phosphorylation of HSP27 during
development and decay of thermotolerance in Chinese hamster cells. J Cell Physiol 147, 93–
101.
Landry J, Lambert H, Zhou M, Lavoie JN, Hickey E, Weber LA & Anderson CW (1992).
Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated
kinases that recognize the same amino acid motif as S6 kinase II. Journal of Biological ….
Lavoie J, Gingras-Breton G, Tanguay R & Landry J (1993). Induction of Chinese hamster
HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization
of the microfilament organization. J Biol Chem 268, 3420–3429.
Lieber R & Thornell L (1996). Muscle cytoskeletal disruption occurs within the first 15 min of
cyclic eccentric contraction. Journal of Applied ….
Holwerda, A - M.Sc Thesis 61
Lieber RL & Fridén J (1999). Mechanisms of muscle injury after eccentric contraction. J Sci
Med Sport 2, 253–265.
Liu Y, Lormes W, Baur C, Opitz-Gress A, Altenburg D, Lehmann M & Steinacker JM (2000).
Human skeletal muscle HSP70 response to physical training depends on exercise intensity.
Int J Sports Med 21, 351–355.
Liu Y, Mayr S, Opitz-Gress A, Zeller C, Lormes W, Baur S, Lehmann M & Steinacker JM
(1999). Human skeletal muscle HSP70 response to training in highly trained rowers. J Appl
Physiol 86, 101–104.
Locke M (2008). Heat shock protein accumulation and heat shock transcription factor activation
in rat skeletal muscle during compensatory hypertrophy. Acta Physiol 192, 403–411.
Locke M, Noble EG & Atkinson BG (1990). Exercising mammals synthesize stress proteins. Am
J Physiol 258, C723–C729.
Locke M, Noble EG & Atkinson BG (1991). Inducible isoform of HSP70 is constitutively
expressed in a muscle fiber type specific pattern. Am J Physiol, Cell Physiol 261, C774–
C779.
Lovering RM & Brooks SV (2013). Eccentric exercise in aging and diseased skeletal muscle:
good or bad? Journal of Applied Physiology; DOI: 10.1152/japplphysiol.00174.2013.
Lowry OH, Rosebrough NJ, FARR AL & Randall RJ (1951). Protein measurement with the
Folin phenol reagent. J Biol Chem 193, 265–275.
Lynn R & Morgan DL (1994). Decline running produces more sarcomeres in rat vastus
intermedius muscle fibres than does incline running. J Appl Physiol 77, 1439–1444.
Lynn R, Talbot JA & Morgan DL (1998). Differences in rat skeletal muscles after incline and
decline running. J Appl Physiol 85, 98–104.
Maglara AA, Vasilaki A, Jackson MJ & McArdle A (2003). Damage to developing mouse
skeletal muscle myotubes in culture: protective effect of heat shock proteins. The Journal of
Physiology 548, 837–846.
Malhotra V & Wong HR (2002). Interactions between the heat shock response and the nuclear
factor-kappaB signaling pathway. Critical Care Medicine 30, S89–S95.
Malm C & Yu J-G (2012). Exercise-induced muscle damage and inflammation: re-evaluation by
proteomics. Histochem Cell Biol; DOI: 10.1007/s00418-012-0946-z.
Malm C, Svensson M, Sjöberg B, Ekblom B & Sjødin B (1996). Supplementation with
ubiquinone-10 causes cellular damage during intense exercise. Acta Physiol Scand 157, 511–
512.
Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM & Dillmann WH (1995). Overexpression
Holwerda, A - M.Sc Thesis 62
of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance
of the heart to ischemic injury. J Clin Invest 95, 1446–1456.
McArdle A, Dillmann WH, Mestril R, Faulkner JA & Jackson MJ (2004). Overexpression of
HSP70 in mouse skeletal muscle protects against muscle damage and age-related muscle
dysfunction. The FASEB Journal 18, 355–357.
McArdle A, Pattwell D, Vasilaki A, Griffiths RD & Jackson MJ (2001). Contractile activity-
induced oxidative stress: cellular origin and adaptive responses. Am J Physiol, Cell Physiol
280, C621–C627.
McHugh MP (2003). Recent advances in the understanding of the repeated bout effect: the
protective effect against muscle damage from a single bout of eccentric exercise. Scand J
Med Sci Sports 13, 88–97.
McHugh MP, Connolly DA, Eston RG & Gleim GW (1999). Exercise-induced muscle damage
and potential mechanisms for the repeated bout effect. Sports Med 27, 157–170.
McHugh MP, Connolly DAJ, Eston RG, Gartman EJ & Gleim GW (2001). Electromyographic
analysis of repeated bouts of eccentric exercise. Journal of Sports Sciences 19, 163–170.
McLoughlin TJ, Mylona E, Hornberger TA, Esser KA & Pizza FX (2003). Inflammatory cells in
rat skeletal muscle are elevated after electrically stimulated contractions. J Appl Physiol 94,
876–882.
McNeil PL & Khakee R (1992). Disruptions of muscle fiber plasma membranes. Role in
exercise-induced damage. The American Journal of Pathology 140, 1097.
Milne KJ & Noble EG (2002). Exercise-induced elevation of HSP70 is intensity dependent. J
Appl Physiol 93, 561–568.
Morgan DL (1990). New insights into the behavior of muscle during active lengthening.
Biophysical Journal 57, 209–221.
Morgan DL & Allen DG (1999). Early events in stretch-induced muscle damage. J Appl Physiol
87, 2007–2015.
Morimoto RI (1998). Regulation of the heat shock transcriptional response: cross talk between a
family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12,
3788–3796.
Morton JP (2006). Time course and differential responses of the major heat shock protein
families in human skeletal muscle following acute nondamaging treadmill exercise. Journal
of Applied Physiology 101, 176–182.
Morton JP, Croft L, Bartlett JD, MacLaren DPM, Reilly T, Evans L, McArdle A & Drust B
(2009a). Reduced carbohydrate availability does not modulate training-induced heat shock
protein adaptations but does upregulate oxidative enzyme activity in human skeletal muscle.
Holwerda, A - M.Sc Thesis 63
Journal of Applied Physiology 106, 1513–1521.
Morton JP, Kayani AC, McArdle A & Drust B (2009b). The exercise-induced stress response of
skeletal muscle, with specific emphasis on humans. Sports Med 39, 643–662.
Morton JP, MacLaren DPM, Cable NT, Campbell IT, Evans L, Bongers T, Griffiths RD, Kayani
AC, McArdle A & Drust B (2007). Elevated core and muscle temperature to levels
comparable to exercise do not increase heat shock protein content of skeletal muscle of
physically active men. Acta Physiol 190, 319–327.
Naito H, Powers SK, Demirel HA, Sugiura T, Dodd SL & Aoki J (2000). Heat stress attenuates
skeletal muscle atrophy in hindlimb-unweighted rats. J Appl Physiol 88, 359–363.
Newham DJ, McPhail G, Mills KR & Edwards RH (1983). Ultrastructural changes after
concentric and eccentric contractions of human muscle. J Neurol Sci 61, 109–122.
Nguyen HX & Tidball JG (2003). Expression of a muscle-specific, nitric oxide synthase
transgene prevents muscle membrane injury and reduces muscle inflammation during
modified muscle use in mice. The Journal of Physiology 550, 347–356.
Noble EG, Milne KJ & Melling CWJ (2008). Heat shock proteins and exercise: a primer. Appl
Physiol Nutr Metab 33, 1050–1075.
Nosaka K & Clarkson PM (1995). Muscle damage following repeated bouts of high force
eccentric exercise. Medicine & Science in Sports & Exercise 27, 1263–1269.
Nosaka K & Clarkson PM (1996). Changes in indicators of inflammation after eccentric exercise
of the elbow flexors. Medicine & Science in Sports & Exercise 28, 953–961.
Nosaka K & Newton M (2002). Repeated eccentric exercise bouts do not exacerbate muscle
damage and repair. The Journal of Strength & Conditioning Research 16, 117–122.
Nosaka K, Sakamoto K, newton M & Sacco P (2001). How long does the protective effect on
eccentric exercise-induced muscle damage last? Medicine & Science in Sports & Exercise
33, 1490–1495.
Nosek TM, Brotto MA, Essig DA, Mestril R, Conover RC, Dillmann WH & Kolbeck RC
(2000). Functional properties of skeletal muscle from transgenic animals with upregulated
heat shock protein 70. Physiol Genomics 4, 25–33.
O'Neill DET, Aubrey FK, Zeldin DA, Michel RN & Noble EG (2006). Slower skeletal muscle
phenotypes are critical for constitutive expression of Hsp70 in overloaded rat plantaris
muscle. J Appl Physiol 100, 981–987.
Ogata T, Oishi Y, Roy RR & Ohmori H (2005). Effects of T3 treatment on HSP72 and
calcineurin content of functionally overloaded rat plantaris muscle. Biochem Biophys Res
Commun 331, 1317–1323.
Holwerda, A - M.Sc Thesis 64
Oishi Y, Ogata T, Ohira Y, Taniguchi K & Roy RR (2005). Calcineurin and heat shock protein
72 in functionally overloaded rat plantaris muscle. Biochem Biophys Res Commun 330, 706–
713.
Patel TJ, Cuizon D, Mathieu-Costello O, Fridén J & Lieber RL (1998). Increased oxidative
capacity does not protect skeletal muscle fibres from eccentric contraction-induced injury.
Am J Physiol 274, R1300–R1308.
Pattwell DM & Jackson MJ (2004). Contraction-induced oxidants as mediators of adaptation and
damage in skeletal muscle. Exerc Sport Sci Rev 32, 14.
Paulsen G, Lauritzen F, Bayer ML, Kalhovde JM, Ugelstad I, Owe SG, Hallen J, Bergersen LH
& Raastad T (2009). Subcellular movement and expression of HSP27, B-crystallin, and
HSP70 after two bouts of eccentric exercise in humans. Journal of Applied Physiology 107,
570–582.
Paulsen G, Vissing K, Kalhovde JM, Ugelstad I, Bayer ML, Kadi F, Schjerling P, Hallen J &
Raastad T (2007). Maximal eccentric exercise induces a rapid accumulation of small heat
shock proteins on myofibrils and a delayed HSP70 response in humans. AJP: Regulatory,
Integrative and Comparative Physiology 293, R844–R853.
Phillips SM, Tipton KD, Aarsland A, Wolf SE & Wolfe RR (1997). Mixed muscle protein
synthesis and breakdown after resistance exercise in humans. Am J Physiol Endocrinol
Metab 273, E99–E107.
Pizza F, Peterson J & Baas J (2004). Neutrophils contribute to muscle injury and impair its
resolution after lengthening contractions in mice - Pizza - 2005 - The Journal of Physiology -
Wiley Online Library. The Journal of Physiology 562, 899–913.
Pizza FX, Davis BH, Henrickson SD, Mitchell JB, Pace JF, Bigelow N, DiLauro P & Naglieri T
(1996). Adaptation to eccentric exercise: effect on CD64 and CD11b/CD18 expression. J
Appl Physiol 80, 47–55.
Pizza FX, McLoughlin TJ, McGregor SJ, Calomeni EP & Gunning WT (2001). Neutrophils
injure cultured skeletal myotubes. Am J Physiol, Cell Physiol 281, C335–C341.
Proske U & Morgan D (2001). Muscle damage from eccentric exercise: mechanism, mechanical
signs, adaptation and clinical applications. The Journal of Physiology 537, 333–345.
Puntschart A, Vogt M, Widmer HR, Hoppeler H & Billeter R (1996). Hsp70 expression in
human skeletal muscle after exercise. Acta Physiol Scand 157, 411–417.
Ritossa F (1962). A new puffing pattern induced by temperature shock and DNP in Drosophila.
Cellular and Molecular Life Sciences 18, 571–573.
Salo DC, Donovan CM & Davies KJA (1991). HSP70 and other possible heat shock or oxidative
stress proteins are induced in skeletal muscle, heart, and liver during exercise. Free Radical
Biology and Medicine 11, 239–246.
Holwerda, A - M.Sc Thesis 65
Sam M, Shah S, Fridén J, Milner DJ, Capetanaki Y & Lieber RL (2000). Desmin knockout
muscles generate lower stress and are less vulnerable to injury compared with wild-type
muscles. Am J Physiol, Cell Physiol 279, C1116–C1122.
Sarge KD, Murphy SP & Morimoto RI (1993). Activation of heat shock gene transcription by
heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and
nuclear localization and can occur in the absence of stress. Mol Cell Biol 13, 1392–1407.
Sayers SP, Knight CA & Clarkson PM (2003). Neuromuscular variables affecting the magnitude
of force loss after eccentric exercise. Journal of Sports Sciences 21, 403–410.
Schmitt E, Gehrmann M, Brunet M, Multhoff G & Garrido C (2006). Intracellular and
extracellular functions of heat shock proteins: repercussions in cancer therapy. Journal of
Leukocyte Biology 81, 15–27.
Schwane JA & Armstrong RB (1983). Effect of training on skeletal muscle injury from downhill
running in rats. J Appl Physiol 55, 969–975.
Skidmore R, Gutierrez JA, Guerriero V & Kregel KC (1995). HSP70 induction during exercise
and heat stress in rats: role of internal temperature. Am J Physiol Regul Integr Comp Physiol
268, R92–R97.
Smith ICH & Newham DJ (2006). Fatigue and functional performance of human biceps muscle
following concentric or eccentric contractions. Journal of Applied Physiology 102, 207–213.
Smith LL (1991). Acute inflammation: the underlying mechanism in delayed onset muscle
soreness? Medicine & Science in Sports & Exercise 23, 542–551.
Thomas JA & Noble EG (1999). Heat shock does not attenuate low-frequency fatigue. Canadian
journal of physiology and pharmacology 77, 64–70.
Thompson HS, Clarkson PM & Scordilis SP (2002). The repeated bout effect and heat shock
proteins: intramuscular HSP27 and HSP70 expression following two bouts of eccentric
exercise in humans. Acta Physiol Scand 174, 47–56.
Thompson HS, Maynard EB, Morales ER & Scordilis SP (2003). Exercise-induced HSP27,
HSP70 and MAPK responses in human skeletal muscle. Acta Physiol Scand 178, 61–72.
Thompson HS, Scordilis SP, Clarkson PM & Lohrer WA (2001). A single bout of eccentric
exercise increases HSP27 and HSC/HSP70 in human skeletal muscle. Acta Physiol Scand
171, 187–193.
Tidball JG (2004). Inflammatory processes in muscle injury and repair. AJP: Regulatory,
Integrative and Comparative Physiology 288, R345–R353.
Tiidus PM (2008). Skeletal muscle damage and repair. Human Kinetics Publishers.
Tissières A, Mitchell HK & Tracy UM (1974). Protein synthesis in salivary glands of Drosophila
Holwerda, A - M.Sc Thesis 66
melanogaster: relation to chromosome puffs. J Mol Biol 84, 389–398.
Touchberry CD, Gupte AA, Bomhoff GL, Graham ZA, Geiger PC & Gallagher PM (2012).
Acute heat stress prior to downhill running may enhance skeletal muscle remodeling. Cell
Stress and Chaperones 17, 693–705.
Towbin H, Staehelin T & Gordon J (1979). Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl
Acad Sci USA 76, 4350–4354.
Tupling AR, Bombardier E, Stewart RD, Vigna C & Aqui AE (2007). Muscle fiber type-specific
response of Hsp70 expression in human quadriceps following acute isometric exercise.
Journal of Applied Physiology 103, 2105–2111.
Vasilaki A, McArdle F, Iwanejko LM & McArdle A (2006). Adaptive responses of mouse
skeletal muscle to contractile activity: The effect of age. Mechanisms of Ageing and
Development 127, 830–839.
Vissing K, Bayer ML, Overgaard K, Schjerling P & Raastad T (2009). Heat shock protein
translocation and expression response is attenuated in response to repeated eccentric
exercise. Acta Physiol 196, 283–293.
Walsh RC, Koukoulas I, Garnham A, Moseley PL, Hargreaves M & Febbraio MA (2001).
Exercise increases serum Hsp72 in humans. Cell Stress and Chaperones 6, 386–393.
Warren GL, Hayes DA, Lowe DA & Armstrong RB (1993). Mechanical factors in the initiation
of eccentric contraction-induced injury in rat soleus muscle.
Warren GL, Hermann KM, Ingalls CP, Masselli MR & Armstrong RB (2000). Decreased EMG
median frequency during a second bout of eccentric contractions. Medicine & Science in
Sports & Exercise 32, 820–829.
Warren GL, Ingalls CP, Lowe DA & Armstrong RB (2001). Excitation-contraction uncoupling:
major role in contraction-induced muscle injury. Exerc Sport Sci Rev 29, 82–87.
Warren GL, Lowe DA & Armstrong RB (1999). Measurement tools used in the study of
eccentric contractioninduced injury. Sports Med 27, 43–59.
Weitzel G, Pilatus U & Rensing L (1985). Similar dose response of heat shock protein synthesis
and intracellular pH change in yeast. Exp Cell Res 159, 252–256.
Welch WJ (1992). Mammalian stress response: cell physiology, structure/function of stress
proteins, and implications for medicine and disease. Physiological Reviews 72, 1063–1081.
Welch WJ, Garrels JI, Thomas GP, Lin JJ & Feramisco JR (1983). Biochemical characterization
of the mammalian stress proteins and identification of two stress proteins as glucose-and
Ca2+-ionophore-regulated proteins. J Biol Chem 258, 7102–7111.
Holwerda, A - M.Sc Thesis 67
Whitehead NP, Weerakkody NS, Gregory JE, Morgan DL & Proske U (2001). Changes in
passive tension of muscle in humans and animals after eccentric exercise. The Journal of
Physiology 533, 593–604.
Zádor E, Mendler L, Takács V, de Bleecker J & Wuytack F (2001). Regenerating soleus and
extensor digitorum longus muscles of the rat show elevated levels of TNF-alpha and its
receptors, TNFR-60 and TNFR-80. Muscle Nerve 24, 1058–1067.
Holwerda, A - M.Sc Thesis 68
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
Holwerda, A - M.Sc Thesis 69
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
Holwerda, A - M.Sc Thesis 70
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.
Holwerda, A - M.Sc Thesis 71
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
11 90% Ethanol Rinse
12 95% 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.
Holwerda, A - M.Sc Thesis 72
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.
Holwerda, A - M.Sc Thesis 73
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 1st wash - TTBS (5min)
o 2nd wash - TTBS (5min)
o Incubate in 2% blotto with secondary anti-body for 1-2h at room temperature
12. 1:2500: 20mL blotto, 8μL antibody
o 1st wash - TTBS (5min)
o 2nd wash - TTBS (5min)
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
Holwerda, A - M.Sc Thesis 74
top
16. Expose for 1 min, 3 min, 5min, choose best one.
o Develop: 5min in Developer, 3min water, 3min Fix, 3min water, Dry.
Holwerda, A - M.Sc Thesis 75
Appendix 2 – Compiled Raw Data
Table 5 - Shortening Contractions
Holwerda, A - M.Sc Thesis 76
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)