The Effect of Estrogen Deprivation on the Cellular Stress

Response Following Muscle Damage

By

Stephanie Salerno

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Exercise Sciences University of Toronto

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The Effect of Estrogen Deprivation on the Cellular Stress Response Following Muscle Damage

Stephanie Salerno

Master of Science

Graduate Department of Exercise Sciences

University of Toronto

2017 Abstract TA muscles from ovary-intact (OVI; n=12) and ovariectomized (OVX; n=12)

female Sprague-Dawley rats were subjected to either 20 or 40 LCs. No differences in

any muscle contractile properties were observed in TA muscles between OVI and

OVX animals. Skeletal muscle fibre changes following ovariectomy included

increased (P<0.001) fibre area in the contra-lateral (control) muscle from OVX

animal and increased (P<0.001) endomysial infiltration score following 20 and 40

LCs from OVX animals. Heat shock protein 25 (Hsp) TA muscle content was

increased (P<0.05; P<0.01, respectively) in TA muscle from OVX animals following

20 and 40 lengthening contractions (LC) and in OVI animals following 40 LCs

(P<0.01). A decreased constitutive expression of Hsp72 (P<0.05) in OVX animals

was observed. Following 40 LCs, TA muscle Hsp72 content was increased in OVI

animals (P<0.05). Thus, estrogen may play a role in regulating skeletal muscle

cellular stress response and damage following controlled LCs.

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Acknowledgements: I would like to extend my utmost gratitude to Dr. Marius Locke for providing me

with the opportunity to take on this research project. His guidance and patience

have helped navigate me through the trials and challenges of my research. Without

his guidance and support I could not have been successful. Thank you greatly for

your encouragement and direction. I would also thank the members of my

committee Dr. Daniel Moore and Dr. Cathy Amara for their support and guidance

throughout my research.

Many thanks to my friends, family, and lab mates who supported me through this

project and all the highs and lows.

My greatest heartfelt thanks to my good friend Dr. Lilia Topouzova, if not for her I

would not have taken on this challenge. I would like to thank her for believing in me

and pushing me to be my very best.

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Table of Contents Abstract ………………………………………………………………………………………………...…….…… ii Acknowledgements ……………………………………………………………………………….……….... iii Table of Contents ………………………………………………………………………………….….……… iv List of Tables ……………………………………………………………………………………………...….. viii List of Figures ……………………………………………………………………………………..…………… iv Abbreviations ………………………………………………………………………………...……………….... x Chapter I: Review of Literature 1.1 Introduction …………………………………………………………………………………..…………… 1 1.2 The Stress of Exercise ………………………………………………………………………..………... 2 1.3 The Structure of Skeletal Muscle ………………………………………………………………….. 2 1.4 Contraction Types ………………………………………………………………..…………………….. 3 1.5 Muscle Damage …………………………………………………………………………..……………….. 3

1.5.1 Consequences of Muscle Damage ………………………………………..…………….. 4 1.6 Possible Mechanisms of Muscle Damage ………………………………..…...……..…………. 7 1.7 Estrogen …………………………………………………………………………………...………………… 7 1.8 Proposed Mechanisms of Estrogen Protection ………………………………...……………. 8

1.8.1 The Antioxidant Properties of Estrogen ……………………………………..……... 9 1.8.2 The Membrane Stabilizing Properties of Estrogen ………………………….... 10

1.9 The Effect of Estrogen Loss or Deprivation on Skeletal Muscle ……………………………………………………………………………………………......………….. 11 1.10 The Loss or Deprivation of Estrogen on Human Skeletal Muscle ………….……. 11 1.11 Heat Shock Proteins ……………………………...………………………………………………… 13 1.12 Heat Shock Protein 25 …………………………………………………………………………...... 14 1.13 Heat Shock Protein 72 ………………………………………………………………………….….. 15 1.14 The Role of Heat Shock Proteins in Skeletal Muscle ………………………...………... 16 1.15 The Effect of Estrogen on Heat Shock Proteins ………................................................. 17 Chapter II: Study Rationale and Objectives 2.1 Study Rationale ……………………………………………………………………………………...…. 19 2.2 Objective ……………………………………………………………………………………………...…… 21 2.3 Hypothesis ……………………………………………………………………………………………...… 21 2.4 Study Question ………………………………………………………………………………………….. 21 Chapter III: Methods and Materials 3.1 Animals ……………………………………………………..……………………………………………… 22 3.2 Stimulation Protocol ………………………………….……………………………………………… 22 3.3 Tissue Collection and Uterus Dissection …..….………………………………………..…… 24 3.4 Biochemical Analysis ………..……………………………………….………………………………. 25

3.4.1 Tissue Homogenization ………………………………….………………………….....… 25 3.4.2 Protein Content Calculation …………………………….…..………………………….. 26 3.4.3 Lowry—Protein Concentrations ……………………….…………………………….. 26 3.4.4 One-Dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis …………………………………………………..……………………………. 27

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3.4.5 Western Blotting—Protein Analysis ……………………..………..…………......... 28 3.4.6 ELISA—Blood Serum Analysis ………………………..………………….…………… 29

3.5 Morphological Analysis …………………………………………………………………..………..... 30 3.5.1 Muscle Cross-Sectioning ………………………………………………………..……….. 30 3.5.2 Hematoxylin and Eosin Staining ……………………………….……………..……… 30 3.5.3 ImageJ Quantification ……………………………………………..………………..…….. 31 3.5.4 Histological Quantification ………………………………………………………....…... 32

3.6 Statistical Analysis …………………………………………………………………………………..… 33 Chapter IV: Results 4.1 Anatomical and Physiological Alterations ……………………………..…..………………... 34

4.1.1 Anatomical Changes Following Ovariectomy ………………………………….... 34 4.1.2 The Effect Lengthening Contractions on TA Muscle Mass ….……..……….. 35 4.1.3 Muscle Protein Concentration …………………………………………..…………..…. 39

4.2 Muscle Contractile Alterations …………………………..………………………….……............ 39 4.2.1 Muscle Contractile Properties ………………………..……………………..…………. 39 4.2.2 Torque Throughout 20 and 40 Lengthening Contractions ……….……….. 41 4.2.3 The Active Component of Tibialis Anterior Muscle Torque ………..…...… 44 4.2.4 The Passive Component of Tibialis Anterior Muscle Torque …...…..……. 48 4.2.5 Maximal Tetanic Tension Prior to and Following 20 and 40 Lengthening Contractions ………………………….………………………....… 52 4.2.6 Maximal Twitch Tension Prior to and Following 20 and 40 Lengthening Contractions ………………………………………………….....… 56

4.3 Muscle Fibre Morphological and Histological Changes Following Ovariectomy and/or Lengthening Contractions …………...………….……...……………….. 60

4.3.1 Histological Measures of Muscle Damage ………………..……………………..... 60 4.3.2 Muscle Fibre Morphology in the Contra-lateral (Control) TA Muscles from OVI and OVX Animals ……..……………………..…..……..………………... 60 4.3.3 Visual Analysis of Muscle Damage from TA Muscles from OVI and OVX Animals Subjected to Lengthening Contractions …….….………….…… 61 4.3.4 TA Muscle Fibre Area from OVI and OVX Animals Following Lengthening Contractions ……………….…………………………. 61 4.3.5 TA Muscle Fibre Circularity from OVI and OVX Animals Following Lengthening Contractions ………………….………………………. 62 4.3.6 TA Muscle Fibre Roundness from OVI and OVX Animals Following Lengthening Contractions …………..……………….…………….. 62 4.3.7 TA Muscle Fibre Necrosis from OVI and OVX Animals Following Lengthening Contractions ………..………………………………... 63 4.3.8 Endomysial Infiltration of TA Muscle Fibre from OVI and OVX Animals Following Lengthening Contractions ……………….………...…. 64 4.3.9 Endomysial Distension of TA Muscle Fibre from OVI and OVX Animals Following Lengthening Contractions ………………….………… 65 4.3.10 Perimysial Infiltration and Distension of TA Muscle Fibres from OVI and OVX Animals Following Lengthening Contractions …... 65

4.4 The Cellular Stress Response Following Ovariectomy and/or Lengthening Contraction …………………………………………………………………….. 71

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4.4.1 The Constitutive Expression of Heat Shock Protein 25 in Tibialis Anterior Muscle ………………………………………………………………….……… 71 4.4.2 The Constitutive Expression of Heat Shock Protein 72 in Tibialis Anterior Muscle ……………………………………………………………………….… 73 4.4.3 TA Muscle Heat Shock Protein 25 Content Following Lengthening Contractions ………………………………………………….…………………… 75 4.4.4 Heat Shock Protein 72 Content in the Lengthening Contracted TA Muscle …………………………………..……..………………….……………… 77 4.4.5 HSF1 Content ……………………………………………………………………….………... 79

4.5 Summary ……………………………………………………………………………………………….…. 81 4.5.1 Anatomical, Physiological and Muscle Contractile Changes Following Ovariectomy and/or Lengthening Contractions ……………..………… 81 4.5.2 The Changes in TA Muscle Fibre Morphology Following Ovariectomy and/or Lengthening Contractions …………………………….………… 81 4.5.3 The Muscle Cellular Stress Response Following Ovariectomy and/or Lengthening Contractions ……………………………………………………….….. 82

Chapter V: Discussion 5.1 Overview …………………………………………………………………..……………………………… 83 5.2 Lengthening Contractions vs. Downhill Treadmill Running ………………..……….. 84 5.3 Skeletal Muscle Contractility …………………………………………………………………..…. 86

5.3.1 Tibialis Anterior Muscle Torque ………………………………………………...…… 86 5.3.2 Maximal Tetanic Tension and Twitch Tension Recovery Following Lengthening Contractions ……………………………………….…………..….. 87 5.3.3 The Active Component of Tibialis Anterior Muscle Torque ………………………………………………………………...……………………... 90 5.3.4 The Passive Component of Tibialis Anterior Muscle Torque …………............................................................................................................ 90

5.4 Histological Characteristics ………………………………………………………………...……... 92 5.4.1 Muscle Fibre Area and Circularity in Ovary-Intact and Ovariectomized Animals ………………………………………………………...………... 92 5.4.2 Endomysial Infiltration …………………………………………………………………... 92 5.4.3 Endomysial Distension Between Ovary-Intact and Ovariectomized Animals ……………………………………………………………...…... 94

5.5 The Cellular Stress Response Following Ovariectomy and/or Lengthening Contractions ……………………………………………………………….…. 95

5.5.1 Differences in Heat Shock Protein 25 and 72 Content Following Lengthening Contractions ………………………………………….. 97 5.5.2 The Constitutive Expression of Heat Shock Protein 72 in the Tibialis Anterior ……………………………………………………………..…………….. 98 5.5.3 Heat Shock Factor 1 Content in Ovariectomized Animals ………….……… 99 5.5.4 Heat Shock Protein 72 Expression in Ovariectomized Animals ………… 99

5.6 Connections to Post-Menopausal Females …………….……………………..…………... 101 5.7 Conclusions ………………………………………………………………………………….…………. 102 5.8 Limitations ……………………………………………………………………….…………………….. 102

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Chapter VI: References ……………………………………………………..………………………… 105 Chapter VII: Appendix 7.1 Tissue Preparation and Sectioning Skeletal Muscle …………………………..………. 122 7.2 Tissue and Blood Collection ……………………………………………………………..……… 124 7.3 Muscle Homogenization ……………………………………………………………………..…… 126 7.4 Determination of Protein Concentration—Lowry Assay ………………………..….. 128 7.5 SDS-PAGE, Western Blotting and Development ……………………………………...…. 129 7.6 Enzyme-Linked Immunosorbent Assay (ELISA) ………………………………..……… 131 7.7 Muscle Cross-Sectioning …………………………………………………………………..……… 132 7.8 Hematoxylin and Eosin Staining ………………………………………………………..……... 134 7.9 Morphological Scoring System …………………………………………………………..…….. 135

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List of Tables Table 1: The Effects of Ovariectomy on Body, Muscle and Uteri Masses, and 17-β Estradiol Concentration …………………………………………….……………………… 37 Table 2: Tibialis Anterior Muscle Mass in the Lengthening Contracted and Contra-lateral (Control) Limbs …………………………….……………..….... 38 Table 3: Skeletal Muscle Fibre Alterations Following 20 and 40 Lengthening Contractions ……………………………….………………. 69 Table 4: Skeletal Muscle Fibre Damage Following 20 and 40 Lengthening Contractions …………………………………..…………… 70

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List of Figures Figure 1: The Active and Passive Component of TA Muscle Torque in Lengthening Contractions ……………………………………………………. 40 Figure 2: Muscle Torque is Similar Between OVI and OVX Animals Throughout 20 Lengthening Contractions …………………………………………………..…… 42 Figure 3: Muscle Torque is Similar Between OVI and OVX Animals Throughout 40 Lengthening Contractions …………………………………………..…………… 43 Figure 4: Ovariectomy Does Not Alter the Active Component of Muscle Torque Throughout 20 Lengthening Contractions ………………………………….………….. 46 Figure 5: Ovariectomy Does Not Alter the Active Component of Muscle Torque Throughout 40 Lengthening Contractions ……………………………………………... 47 Figure 6: Ovariectomized Animals Maintained the Passive Component of Torque Throughout 20 Lengthening Contractions ………………………...……………… 50 Figure 7: Ovariectomized Animals Maintained the Passive Component of Torque Throughout 40 Lengthening Contractions …………………………………….….. 51 Figure 8: Maximal Tetanic Tension is Similar Between TA Muscles from OVI and OVX Animals Following 20 Lengthening Contractions ……………………………...... 54 Figure 9: Maximal Tetanic Tension is Decreased Following 40 Lengthening Contractions ……………………………………………………….…….………………… 55 Figure 10: Maximal Twitch Tension is Decreased Following 20 Lengthening Contractions ………………………………………………………………….……….….. 58 Figure 11: Maximal Twitch Tension is Decreased Following 40 Lengthening Contractions …………………………………………………………………….….……… 59 Figure 12: Skeletal Muscle Changes in Ovary-Intact and Ovariectomized Animals Following 20 or 40 Lengthening Contractions …………………………….………. 66 Figure 13: Skeletal Muscle Damage Following Lengthening Contractions in Ovary-Intact Animals …………………..…………………………………………………..…….……. 67 Figure 14: Skeletal Muscle Damage Following Lengthening Contractions in Ovariectomized Animals ……………………………………………………………..……..……….. 68 Figure 15: Constitutive Hsp25 Content is Unchanged Following Ovariectomy …………………………………………………………………………….…….. 72 Figure 16: Constitutive TA Muscle Hsp72 Expression is Significantly Decreased Following Ovariectomy ……………………………………………………………………..………….… 74 Figure 17: TA Muscle Hsp25 Content Increases Following Lengthening Contractions ……………….………………………………………………………...……. 76 Figure 18: TA Muscle Hsp72 Content Following 20 and 40 Lengthening Contractions in Ovary-Intact and Ovariectomized Animals …………………………...….. 78 Figure 19: Heat Shock Factor1 Content in TA Muscles from OVX Animals …………………………………………………………………………………………………... 80

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Abbreviations Ca2+—Calcium CC—Control-Control CK—Creatine Kinase CSR—Cellular Stress Response DAMPs—Damage Associated Molecular Patterns EDL—Extensor Digitorum Longus ED1+--Subpopulation of macrophages (pro-inflammatory) ED2+--Subpopulation of macrophages (anti-inflammatory) ELISA—Enzyme-linked Immunosorbent Assay FOXO3A—Foxhead Box O3 G—Gauge g—gram H&E—Hematoxylin and Eosin HSP—Heat Shock Protein kDA—Kilodalton LC—Lengthening Contractions mg—Milligram ml—Milliliter MTT—Maximal Tetanic Tension MTw—Maximal Twitch Tension NFκB—Nuclear Factor-κ-Beta OVI—Ovary-intact OVX—Ovariectomized ROS—Reactive Oxygen Species SC—Shortening Contraction TA—Tibialis Anterior TNF-α—Tumor Necrosis Factor-α w/v—Weight per volume

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Chapter I: Review of Literature 1.1 Introduction

Hans Selye first characterized the body’s response to life’s challenges in the

mid 20th century. During periods of stress, the body exhibits a series of complex and

powerful responses that allows one to cope with the stressful condition/state

(Nickerson, 2006). Originally termed the ‘alarm response,’ stress was first

characterized as a general phenomenon (Selye, 1956; Tache and Selye, 1985).

Although stressors can affect the human body in a global manner, these effects can

also be observed within various systems or tissues including skeletal muscle. Daily

stressors, such as exercise, can impose physical demands activating the ‘alarm

response’ and other reactions responses in both muscle and the body. The goal of

the response is restore homeostasis.

With increased age both males and females are negatively affected by the

decline in skeletal muscle mass and strength. Termed sarcopenia, more than 30% of

individuals over 60 years of age are negatively impacted (Velders and Diel, 2013).

The age-related loss of skeletal muscle is thought to be a multi-factorial process

composed of numerous events, including inactivity, oxidative stress, nutritional

intake, inflammatory status and hormonal changes (Abbasi et al., 1998;

Baumgartner et al., 1999; Roubenoff and Hughes, 2000). The consequence of a

decreased muscle mass is the resultant increased health risks and decreased quality

of life.

In females, the presence of estrogen has been shown to provide some

resistance to cellular damage from certain pathological conditions such as

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cardiovascular disease, Parkinson’s disease and aging-related dementia

(Mendelsohn and Karas, 1999; Garcia-Segura et al., 2001; Brinton, 2004). In the

developed world, most females will spend one-third of their lifetime in a state of

estrogen deprivation (Barrett-Connor, 1993). Given that the risk for sarcopenia

increases with age, it suggests estrogen may play a role in protecting skeletal

muscle, thus emphasizing its importance.

1.2 The Stress of Exercise

Exercise can be a physical stress that ultimately results in cellular and

systemic adaptations. This may happen through two ways: the first is through

hypothalamic-pituitary axis, which is responsible for controlling and regulating

many vital bodily processes (Caston et al., 1995). The second involves hormones,

including estrogen, that bind to membrane receptors, move to the nucleus and alter

gene expression (Pinilla et al., 1992; Jarow et al., 1993; Chennaoui et al., 2002;

Vicdan, 2006). Both ways work together to cope with the stress of exercise (Viau,

2002) and restore homeostasis following exercise.

1.3 The Structure of Skeletal Muscle

Skeletal muscle is comprised of myofibres consisting primarily of actin and

myosin. The sarcomere is the functional unit of skeletal muscle and produces force

by actin and myosin cross bridge interaction. Skeletal muscle is comprised of

multiple bundles of cells known as muscle fascicles, surrounded by a layer of

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connective tissue. Each muscle fascicle is comprised of muscle fibres, which in turn

are constituted by myofibrils.

1.4 Contraction Types

Skeletal muscle contractions can be separated into isometric or dynamic

contractions. An isometric contraction results in no change in muscle length while a

dynamic contraction changes muscle length such that it shortens (concentric) or

lengthens (eccentric). With shortening contractions the collective forces generated

by the muscle fibres are greater than the opposing load and the sarcomeres shorten.

In contrast, a LC occurs when the muscle forces generated are insufficient to move

the load and the muscle is forcibly lengthened. For example, during a squat when

the load is lifted (up) the quadriceps performs a shortening contraction (SC).

Conversely, when the load is lowered (against the force of gravity) the quadriceps

undergo a LC. In comparison to SCs, LCs is associated with greater subsequent

muscle fibre damage and hence greater torque loss (Fridén et al. 1983; Evans et al.

1985; Fridén and Lieber, 1992).

1.5 Muscle Damage

As mentioned previously, skeletal muscle has evolved to shorten. However,

when the load is greater than the force, sarcomeres lengthen. As a result of LCs the

sarcolemma, sarcoplasmic reticulum and z-lines can become damaged, resulting in

protein leakage (Stupka et al., 2000). Following these primary events in muscle

damage, LC biased exercise results in the elevation of oxygen free radicals, further

4

propagating a secondary damage. The most noticeable consequences of muscle fibre

damage following LC exercise include delayed onset muscle soreness and an

immediate immune response (Stupka et al., 2000). These alterations may last up to

ten days following exercise (Tiidus, 2000).

1.5.1 Consequences of Muscle Damage Swelling

Muscle swelling or fluid accumulation often occurs following damaging

exercise and results in an increased muscle mass (McCully and Faulkner, 1985). An

increased muscle mass of approximately 10% has been observed immediately after

(t=0) forced LCs (Komulainen et al., 1998). Fibre swelling generally peaks 4-5 days

following muscle injury and gradually spreads throughout the muscle (Yu et al.,

2013). Fluid accumulation may remain in the intracellular space for as long as 5

days following damaging exercise (Yu et al., 2013).

Torque Loss

Following a sufficient stimulus of either SC or LCs, any subsequent

contractions may show a decreased torque. However, following SCs, the causes of

reduced muscle torque are generally due to metabolic fatigue. Indeed, SCs have been

shown to result in torque losses up to 30%, although torque generally returns

within hours following exercise (Newham et al., 1983; Jones et al., 1989). In

contrast, LCs may cause torque losses up to 60% (Newham et al., 1987, Nosaka et al.,

1991; Saxton et al., 1995) and depending on the severity, may persist for weeks

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(Newham et al., 1987). This long-term torque loss is thought to be due to the

damage to structural or cytoskeletal components that are associated with LCs

(Semmler, 2014). For example, central nuclei (Faulkner et al., 1989), z-line

streaming (Brooks et al., 1995) and ghost fibres (Komulainen et al., 1998; Holwerda

and Locke, 2014) are commonly observed following LCs. Thus, while both SCs and

LCs result in a decreased torque, a more persistent and greater magnitude of torque

loss is associated with LCs.

Leukocyte Infiltration

Following exercise induced muscle damage, leukocytes (neutrophils and

macrophages) migrate to the site of injury (Peake et al., 2005). The time-course of

this inflammatory response is dependent on the mode, duration, intensity and

muscle groups utilized during exercise (Smith et al., 1989; MacIntyre et al., 1995).

Within hours following LC, neutrophils invade the injured muscle fibres (MacIntyre

et al., 1996; MacIntyre et al., 2000; Malm et al., 2000; Stupka et al., 2000; Beaton et

al., 2002; Raastad et al., 2003) and may remain for up to 5 days (Yu et al., 2013).

High concentrations of neutrophils are required for proteolysis in order to remove

cellular debris, which are common signs of fibre damage and degeneration (Tidball,

2005). Following the initial fibre damage, neutrophils release proteolytic enzymes,

such as calpains, and oxygen free radicals, which can result in a further tissue

(secondary) degradation (Cannon et al., 1990; Pizza et al., 1995; Clarkson and Hubal,

2001). This allows for a greater efflux of muscle proteins such as creatine kinase

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(CK) (Cannon et al., 1990; Pizza et al., 1995), or even heat shock proteins (Hsp)

(Zheng et al., 2004).

Following muscle damage and the invasion of neutrophils, macrophages

typically enter the fibre 12 to 48 hours later (Tidball et al., 1995; Tiidus et al., 2001).

Two subpopulations of macrophages, termed ED1+ and ED2+ have been identified

(Honda et al., 1990; St. Pierre and Tidball, 1994). Approximately 12 hours following

damaging exercise, ED1+ macrophages invade (Clarkson and Sayer, 1999) whereas

ED2+ macrophages enter the fibre between 24 to 48 hours after exercise. ED1+

macrophages act as phagocytes and remove cellular debris in necrotic tissue (St.

Pierre and Tidball, 1994; Tidball, 2005) whereas ED2+ cells are necessary for

myoblast proliferation in muscle recovery (St. Pierre and Tidball, 1994; Massimino

et al., 1997; Tidball, 2005) and are thought to regulate the repair process (Clarkson

and Sayer, 1999). Thus, the increase in leukocyte infiltration following muscle

damage may be used as a measure or marker of muscle damage.

Neutrophil and macrophage infiltration and cytokine expression are

sensitive to ovarian hormone expression (Zuckerman et al., 1996; Angstwrum et al.,

1997; Komulainen et al., 1999; Tiidus et al., 2001, 2005). In support of this, female

rodents subjected to LCs displayed decreased macrophage invasion when compared

to males (St. Pierre-Schneider et al., 1999). Similarly, McClung et al. (2007) showed

the soleus muscle of OVX rats displayed elevated neutrophil and macrophage

concentrations following recovery from disuse injury. This work suggests that

estrogen deprived females may exhibit a greater inflammatory response following

exercise, which may lead to a delayed or decreased ability to recover.

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1.6 Possible Mechanisms of Muscle Damage Two mechanisms have been proposed to explain the decrement in torque

following exercise-induced muscle damage. The first is the “popping sarcomere”

hypothesis proposed by Morgan (1990). Also known as sarcomere disorganization,

this damage occurs when the muscle is stretched beyond optimal length pulling the

weakest sarcomeres (longest sarcomere) apart. These sarcomeres become

progressively weaker until the rising passive tension compensates for the

decreasing active tension (Morgan and Poske, 2004). This results in the sarcomeres

being stretched past the point of myofilament overlap and “popping” out (Morgan,

1990) thereby damaging the sarcomeres and decreasing muscle torque.

The second proposed mechanism for muscle damage following exercise is

the disruption of calcium (Ca2+) homeostasis. Lengthening contractions cause

membrane damage to the sarcolemma (Takekura et al., 2001) resulting in elevated

resting Ca2+ levels from an inward movement (Morgan and Allen, 1999). The

disrupted muscle sarcolemma is likely due to increased Ca2+ influx, which may also

disrupt the excitation-contraction coupling process (Jones, 1996; Ingalls et al., 1998)

and decreased muscle torque (Overgaard et al., 2002; 2004). Thus, current evidence

suggest LC damage decreases torque and may be the result of over-stretched

sarcomeres and/or disrupted Ca2+ homeostasis.

1.7 Estrogen

In eumenorrheic females, estrogen exists in three forms: the major form of

17-β estradiol and lesser quantities of estrone and estriol (Kirilovas et al., 2007). For

8

ease of communication in this thesis these forms will collectively be referred to as

estrogen. Primarily produced in the ovaries, estrogen is also synthesized in low

amounts in other tissue such as bone and skeletal muscle (Nelson and Bulun, 2001;

Chowdhury and Pickering, 2006; Aizawa et al., 2008). Estrogen is a pleiotropic

hormone and affects muscle function both directly as well as through the central

nervous system (McEwen and Alves, 1999). Estrogen replacement has beneficial

effects on bone, neurons, the cardiovascular system (Persky et al., 2000; Jia et al.,

2008) and skeletal muscle, thus attesting to its importance in musculoskeletal

health.

1.8 Proposed Mechanism of Estrogen Protection

Female sex hormones (estrogen and progesterone) have been shown to

enhance muscle mass, strength and attenuate muscle damage following LCs (Velders

and Diel, 2013). When subjected to exercise of a damaging nature such as LC,

ovariectomized animal displayed increased sarcolemma damage, increased blood

CK (Bar et al., 1985; Amelink and Bar, 1986) and an increased inflammatory

response (St. Pierre-Schneider et al., 1999). Thus, estrogen appears to play a role in

attenuating muscle damage.

Three mechanisms have been proposed to explain the protective effects of

estrogen on muscle. First, similar to vitamin E, estrogen may act as an antioxidant,

thereby inhibiting or decreasing membrane peroxidation (Nedergaard et al., 2013).

Second, estrogen may stabilize membrane integrity (McNulty et al., 2000) thereby

decreasing membrane disruption. Third, estrogen is known to regulate Hsp

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expression, thereby providing resistance against muscle damage (Bombardier et al.,

2009). Thus, estrogen may act through multiple mechanisms to provide protection

to skeletal muscle from the stress of exercise.

1.8.1 The Antioxidant Properties of Estrogen

Antioxidant membrane-stabilizing molecules are known to reduce

membrane fluidity (Wiseman et al., 1993; Behl et al., 1997). Estrogen is similar in

function to vitamin E in its chain-breaking action (Sugioka et al., 1987) and thus may

act as an antioxidant (Bar and Amelink, 1997; Tiidus, 1995; Whiting et al., 2000).

Studies have shown estrogen provides physiological protection to rat skeletal

muscle against free radical-mediated lipid peroxidation (Persky et al., 2000; Stupka

and Tiidus, 2001). This may be accomplished through the donation of a hydrogen

from its hydroxyl group to peroxyradicals (Carter et al., 2001). Thus, estrogen has

the ability to scavenge free radicals, thereby minimizing oxidative damage (Sugioka

et al., 1987; Subbiah et al., 1993; Strehlow et al., 2003). The conferred increase in

antioxidant ability via estrogen may promote muscle fibre survival following

damaging exercise.

Due to the structure of estrogens, it is plausible that estrogen may act to limit

free radical accumulation. It is not surprising that the removal of estrogen results in

a decreased ability to negate ROS production. In support if this, OVX rodents show a

decreased myocardial superoxide dismutase activity and expression (Barp et al.,

2002). Thus, estrogen deprivation may result in an increase in muscle fibre damage

10

due to the inability to negate the negative effects of increased ROS produced during

or following exercise.

1.8.2 The Membrane Stabilizing Properties of Estrogen

Membrane disruption is thought to be a key early event following skeletal

muscle damage (Kendall and Eston, 2002). Estrogen may prevent membrane

disruption through its receptor-mediated system (Tiidus, 1999; Tiidus, 2001) or by

directly interacting with the phospholipid membrane, thereby optimizing

membrane fluidity (Tiidus, 1999; Tiidus, 2003). Further support for the importance

of estrogen can be gleaned from the fact that following exercise, OVX female rats

supplemented with estrogen showed a reduction in blood CK (Bar et al., 1988;

Amelink et al., 1990; Persky et al., 2000; Tiidus et al., 2001; Feng et al., 2004). Thus,

estrogen appears to play a role in minimizing membrane disruption/damage

following exercise.

Estrogen also appears to protect muscle fibres by minimizing leukocyte

infiltration since neutrophil infiltration was attenuated by 1-2 hours following

exercise (Tiidus et al., 2001; Tiidus, 2003). Tiidus et al. (2001) observed that

skeletal muscle from estrogen-supplemented rodents showed an attenuated calpain

activity and neutrophil infiltration following downhill treadmill running. Overall,

estrogen appears to influence membrane stabilization, thus decreasing both CK

leakage and leukocyte infiltration following exercise.

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1.9 The Effect of Estrogen Loss or Deprivation on Skeletal Muscle

In female mice, estrogen has been shown to influence the development of

skeletal muscle size (Sciote et al., 2001). Multiple studies have also observed

estrogen affects muscle fatigue and twitch characteristics, including peak tetanic

tension and half-relaxation time (Hatae, 2001; McCormick et al., 2004; Schneider et

al., 2004; Enns and Tiidus, 2010). Thus, estrogen appears to play a role influencing

contractile measures of skeletal muscle.

Skeletal muscles from female rats showed a decreased loss of desmin

(Komulainen et al., 1999), a structural protein that contributes to z-discs alignment

and aids in tension transmission (Morgan and Allen, 1999). Ovariectomized rats

displayed a greater loss of desmin when compared to estrogen-supplemented rats

following treadmill running (Bar et al., 1988; Enns and Tiidus, 2010). Thus, the loss

of estrogen may predispose skeletal muscle to related z-disc damage. Taken

together, these data suggest estrogen may be necessary for the maintenance of

muscle fibre structure or structural proteins, thereby maintaining torque.

1.10 The Loss or Deprivation of Estrogen on Human Skeletal Muscle

Menopause is an age-related condition of female hypogonadism,

characterized by ovarian failure and exemplified by a rapid yet dramatic decline in

female sex hormone production including estrogen (Rannevik et al., 1986). During

menopause, females experience an accelerated loss of muscle mass (Aloia et al.,

1991). Rolland et al. (2007) showed each year following menopause, females lose

approximately 0.6% of muscle mass. This suggests estrogen promotes the

12

maintenance of muscle mass and that estrogen deprivation may hasten the onset of

sarcopenia. The declining estrogen concentrations following menopause may create

a pro-inflammatory state by increasing cytokines such as tumor necrosis factor-

alpha (TNF-α) (Roubenoff, 2003; Messier et al., 2011) and interleuklin-6 (Roubenoff,

2003). Not surprisingly, a link has been proposed between low estrogen

concentrations and decreased protein synthesis (Messier et al., 2011).

The large change in occurrence of “moderate sarcopenia” in females aged 40

to 49 years suggests the prevalence of sarcopenia increases at a time when

significant hormonal changes occur (Messier et al., 2011). In support of this, Jubrias

et al. (1997) reported post-menopausal females had twice the amount of non-

contractile muscle tissue, including intramuscular fat, when compared to younger

menstruating females. As age progresses beyond menopause, a decreased whole

body, trunk and lower extremity lean body mass, concurrent with an increase in

total body and visceral fat has been observed (Svendsen et al., 1995; Poehlman et al.,

1995; Pantopoulos et al., 1996; Douchi et al., 1998; Gambacciani et al., 1999; Douchi

et al., 2002; Lynch et al., 2002; Van Pelt et al., 2002). An increased adiposity (Phillips

et al., 1993; Poehlman et al., 1995; Aloia et al., 1991; Aloia et al., 1995; Douchi et al.,

1998; Poehlman and Tchernof, 1998) and a decreased bone mineral density also

occur (Carr, 2003). The decline in muscle performance following menopause may be

due to altered muscle contractile properties that result from the changes in

hormone concentrations (Pette and Stratin, 2001). In view of this, estrogen appears

to be necessary to maintain both muscle quantity and quality.

13

1.11 Heat Shock Proteins

Heat shock proteins are rapidly inducible, highly conserved proteins

(Kingston et al., 1987; Welch 1992) known to protect cells from stressors, such as

increased temperature (Landry et al., 1982; Tiidus, 1995; Bar and Amelink, 1997),

oxidative stress (Tiidus, 1995; Bar and Amelink, 1997; Bombardier et al., 2013),

decreased pH (Cohen et al., 1991) and reduced fuel (Febbraio et al., 2002). In the

unstressed cell, Hsps generally remain at low levels, but rapidly accumulate in

response to protein damaging stressors (Chiang et al., 1982; Hochstrasser, 1992;

Ellis and Heart, 1996). Hsps have been shown to minimize the aggregation of

unfolded proteins thereby preventing or limiting the detrimental consequences of

protein aggregation (Haslbeck and Viering, 2015). Given that many diseases of aging

result in protein aggregation, Hsps may play a vital role in maintaining or

preventing age-related diseases such as sarcopenia.

Hsps are known to facilitate protein chaperoning, folding, transport, as well

as the removal of damaged proteins (Lindquist and Craig, 1988; Welch, 1992). In

skeletal muscle, Hsps act as both chaperones and as an indicator of stress (Liu and

Steinacker, 2001). By interacting with damaged proteins, Hsps maintain

protetostasis (Liu and Steinacker, 2001). Thus, Hsps serve as both markers of

stress/damage, as well as preventing protein related problems. Both acute (Locke et

al., 1990; Salo et al., 1991; Skidmore et al., 1995; Hernando and Manso, 1997;

McArdle et al., 2001; Milne and Noble, 2002) and chronic (Gonzalez et al., 2000;

Mattson et al., 2000; Naito et al., 2000; Samelan, 2000) exercise has been shown to

14

increase Hsp content in skeletal muscle. Thus, any exercise mediated increase in

Hsps may play a key role in minimizing sarcopenia.

In addition to the Hsp protein chaperone related function, Hsps may also

provide protection to skeletal muscle through anti-inflammatory and apoptotic

mechanisms (Knowlton, 1995; Mosser and Morimoto, 2000; Christians et al., 2002).

Any inability to elevate Hsp content may result in increased muscle damage and

ultimately exacerbate muscle loss in the long-term. Thus, it is possible that estrogen

deficiency alters the skeletal muscle stress response, thereby rendering skeletal

muscle more susceptible to damage. This may increase muscle loss and have

detrimental effects for certain populations of females.

The regulation of Hsps is controlled through heat shock protein factors

(HSFs). In response to protein damaging stressors, the intracellular accumulation of

damaged proteins removes Hsps from heat shock factor 1 (HSF1), thus allowing

HSF1 trimerization (Chiang et al., 1982; Hochstrasser, 1992; Ellis and Heart, 1996).

This allows HSF1 to bind to the heat shock element-DNA (HSE) and initiate Hsp

transcription (Mosser et al., 1993). The subsequent increase in Hsps eventually

results in sequestration of HSF1, thereby ending the Hsp response (Kim et al., 1995;

Marber et al., 1995).

1.12 Heat Shock Protein 25

Heat shock proteins are grouped according to molecular weight. Small Hsps

(Hsp25, also known as Hsp27 in humans) are comprised of a family of nine proteins

sharing a highly conserved α-crystallin domain (Koh, 2002). Small Hsps are

15

molecular chaperones functioning independently of ATP and involved in a number

of cellular functions, including; modulating the cytoskeletal network, preventing

protein aggregation, regulating caspase activity and maintaining redox status

(Brinkmeier and Ohlendieck, 2014). Hsp25 is named due to its molecular mass of

25-kDa. In unstressed skeletal muscle fibres, Hsp25 is at low concentrations in the

cytosol. However, following stress, Hsp25 interacts with cytoskeleton and

myofilaments (Vissing et al. 2009). With the stress of LCs, Hsp25 binds to z-disk-

related structures (Koh, 2002) and possibly functions as a cytoskeletal protective

protein (Vasconsuelo et al., 2010). This may prevent cytoskeletal disruption during

high-force exercise (Koh, 2002). Using a C2C12 cell line, Vasconsuelo et al. (2010)

showed estrogen administration resulted in a time-dependent (5-60 minute)

increase in Hsp25 content. During contraction-induced muscle damage, Hsp25 has

been shown to interact with the myofilaments (Lutsch et al., 1997) and cytoskeleton

(Koh, 2002, 2004; Vissing et al., 2009) as well as with proteins involved with the

cytoskeletal structures (desmin). Thus, following exercise Hsp25 appears to attempt

to minimize cytoskeletal disruption.

1.13 Heat Shock Protein 72 Hsp70 refers to the markers of the 70-kDa family of proteins and consists of

four major Hsp70 isoforms have been characterized (Noble et al., 2008). Hsp78 is

the largest Hsp70 isoform and exists primarily in the endoplasmic and sarcoplasmic

reticulum, while Hsp75 is a mitochondrial import protein (Lui and Steinacker, 2001;

Noble et al., 2008). A 70kDa heat shock cognate (Hsc70 or Hsp73) is constitutively

16

expressed in a wide variety of cells; however, the main stress inducible form of

Hsp70 isoforms is Hsp72 (HspA1A) (Gabai et al., 1998; Yenari et al., 1998; Lui and

Steinacker, 2001; Bidmon et al., 2004; Noble et al., 2008). Locke et al. (1991) have

shown muscles containing type I muscle fibres constitutively express Hsp72,

possibly explaining why type I muscle fibres may be less susceptible to damage LC

biased exercise. Following LCs, Hsp72 accumulates in skeletal muscle (Thompson et

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

Paulsen et al., 2007; Morton et al., 2009; Paulsen et al., 2009; Holwerda and Locke,

2014). McArdle et al. (2004) observed that overexpression of Hsp70 in extensor

digitorum longus (EDL) from mice appeared to provide protection against LCs.

Hsp72 may be important in regulating specific atrophy signaling pathways

(Senf et al., 2008; Senf et al., 2010). Hsp72 has been shown to inhibit the

transcriptional activity of FOXO3a and nuclear factor-kappa beta (NF-κB), thereby

minimizing skeletal muscle atrophy (Senf et al., 2008; Senf et al., 2010). Thus,

following exercise, Hsp72 accumulation may play a key role in regulating atrophy

pathways thereby influencing muscle mass.

1.14 The Role of Heat Shock Proteins in Skeletal Muscle Hsps maintain protein synthesis and/or decrease the rate of muscle protein

breakdown, thereby perhaps slowing disuse muscle atrophy (Miyabara et al., 2012).

Hsps may also be involved in blunting the NF-κB pro-inflammatory pathway (Chen

et al., 2004b, 2004a; Chen and Currie, 2006), by inhibiting the degradation of IκB

complex and thus the subsequent activation of NF-κB (Noble, 2002). In view of this,

17

an increase in Hsps or the ability to increase Hsps may help protect or reduce

skeletal muscle damage by possibly negating the effect of pro-inflammatory

pathways.

Increased levels of Hsp27 and Hsp70 have been reported in human skeletal

muscle following high-force “eccentric exercise” (Reichsman et al., 1991; Thompson

and Scordilis, 1994; Febbraio and Kokoulas, 2000; Feasson et al., 2002; Thompson

et al., 2002; Thompson et al., 2003; Willoughby et al., 2003) and Hsps appear to be

involved in the remodeling and adaptation process (Feasson et al., 2002; Thompson

et al., 2003). In rodent models, during periods of overload stress, (resistance

exercise) Hsps are up-regulated (Oishi et al., 2005; Ogeta et al., 2005; O’Niell et al.,

2006; Paulsen et al., 2007). In addition, following muscle fibre damage, Hsps have

been shown to migrate to contractile protein structures, possibly in an attempt to

provide stabilization (Koh, 2004; Paulsen et al., 2007). An inverse relationship

between CK release and Hsp70 expression following exercise has also been reported

(Liu et al., 1999). Thus, Hsps appear to be initially involved following damaging

exercise (LC) and/or muscle contractions.

1.15 The Effects of Estrogen on Heat Shock Protein Content

Males and females present different profiles of muscle damage following

unaccustomed exercise (Paroo et al., 2002). Estrogen has been shown to decrease

post-exercise skeletal muscle damage and may be a factor involved in the

differences observed in post-exercise Hsp72 levels between males and females

(Paroo et al., 1999). Paroo et al. (2002) observed an estrogen mediated sex-specific

18

Hsp70 response following downhill treadmill running. Males and ovariectomized

female rodents demonstrated a greater Hsp70 response when compared to ovary-

intact females (Paroo et al., 1999; Gillum, 2010). Thus, estrogen may account for the

differences observed in Hsp content following exercise. In skeletal muscle,

Bombardier et al. (2009; 2013) showed estrogen regulates both basal and exercise-

stimulated Hsp70 content. Thus, estrogen appears to be a factor in mediating both

the constitutive expression and stress-induced expression of Hsps.

Myocytes from old ovariectomized rats showed an increased NF-κB activation,

cytokine expression and decreased ability to handle ROS (Stice et al., 2011). The loss

of Hsp content due to aging is thought to be the result of the loss of HSF1s ability to

bind to DNA inducing transcription (Heydari et al., 2000; Lee et al., 2009). Given the

available evidence, the loss of estrogen may play a role in the stress response of

muscle fibres and may augment protein damage from stressors such as LCs.

Studies have demonstrated estrogen-dependent activation of Hsp70 occurs

via the sequential stimulation of NF-κB and HSF1 (Hamilton et al., 2004; Stice and

Knowlton, 2008). The ability of estrogen to up-regulate Hsp72 content in skeletal

muscle may also provide increased protection by limiting the inflammatory

response following exercise (treadmill running) (Bombardier et al., 2009). Overall

these studies suggest estrogen may play a role protecting skeletal muscle from

exercise-induced damage by enhancing or maintaining the Hsp response. Ultimately,

less muscle damage overtime may result in a slowing of sarcopenia and an increased

quality of life.

19

Chapter II: Study Rationale and Objectives

2.1 Study Rationale

A connection between estrogen and the maintenance of muscle health has

been established. When skeletal muscle deprived of estrogen is subjected to

stressors, greater damage and inflammatory response, concurrent with an increased

Hsp content has been observed. Bombardier et al. (2013) found estrogen regulates

both the unstressed and exercise-stimulated Hsp response, suggesting both may be

altered following ovariectomy. This suggests estrogen deprivation may account for a

decreased capacity to protect skeletal muscle following LC exercise.

Downhill treadmill running as a form of exercise has been used to elucidate

the effects of estrogen on skeletal muscle damage (Paroo et al., 1999; Tiidus and

Bombardier, 1999; Tiidus et al., 2001; Bombardier et al., 2009, 2013). However, a

limitation of running models is the inability to control the specific response that

results from muscle contractions. Thus, the exact stressor responsible for the

increase in Hsp content remains unknown. An increase in body temperature

(Landry et al., 1982; Tiidus, 1995; Bar and Amelink, 1997), oxidative stress (Tiidus,

1995; Bar and Amelink, 1997; Bombardier et al., 2013), decreased pH (Cohen et al.,

1991) and decreased fuels (Febbraio et al., 2002) are all capable of inducing a stress

response. Given the vast number of factors capable of increasing protein damage, a

more controlled method, such as electrically stimulated LCs, may allow for a more

precise cause and effect relationship between estrogen deficiency and muscle

damage to be established.

20

A lowered Hsp content in the unstressed muscle from ovariectomized

animals may result in an inability to increase Hsp expression following LCs,

therefore increasing the extent of muscle damage. Using maximal electrically

stimulated LCs to induce muscle damage, this study intended to clarify the

relationship between estrogen deprivation and Hsp content and muscle damage.

By examining and assessing Hsp content and muscle damage following

damaging exercise (LCs) in estrogen deprived and intact states, the role of the CSR

can be assessed following LCs in skeletal muscle. To do this, one hind limb from OVX

and OVI animals was subjected to maximal electrical stimulation. Physiological (TA

muscle torque, maximal tetanic tension and twitch), biochemical (Hsp25, Hsp72 and

HSF1) and morphological (fibre area, circularity, roundess, fibre necrosis,

endomysial infiltration and distension following skeletal muscle cross-sectioning

and staining using hematoxylin and eosin) parameters following of the muscle were

analyzed.

Two different contraction repetitions (20 and 40) were selected to glean the

extent of differences in structural damage. Holwerda and Locke (2014) found large

amounts of structural damage eight hours following 100 LCs. However, to ensure a

range of muscle damage between OVI and OVX observed was the result of estrogen

loss and not a time dependent effect of LC overload, more moderate contraction

repetitions were selected. Pollock-Tahiri and Locke (2016) have observed increased

Hsp25 and 72 content and decreased torque throughout 20 contractions, with little

structural damage. Thus, to determine the role of ovariectomy in structural muscle

damage, 40 LCs was also selected.

21

2.2 Objective To characterize the CSR and muscle damage in TA skeletal muscle from

ovariectomized and ovary-intact animals following maximally stimulated

lengthening contractions.

3.3 Hypotheses

1) Muscles from OVX animals subjected to 20 or 40 LCs will show an increased

CSR response and muscle damage.

2) Following LCs, TA muscles from OVX animals will show an increased

physiological (contractile), biochemical (Hsps), and morphological markers

of skeletal muscle damage.

3.4 Study Question Does ovariectomy (estrogen deficiency) alter the cellular stress response following

electrically stimulated lengthening contractions?

22

Chapter III: Methods and Materials 3.1 Animals

At approximately two months of age, female Sprague-Dawley rats (n=12)

(Charles River Laboratories, Quebec, Canada) underwent surgical removal of the

ovaries (performed by Charles River Laboratories). Ovariectomized and ovary-

intact animals (N=29; consisting of 12 OVX, 12 OVI, 5 control-control [CC] animals

not subjected to any lengthening muscle contractions) were housed in the Ramsey

Wright laboratory (University of Toronto), two per cage with a 12-hour light-dark

cycle. Animals were fed and provided water ad libitum. All procedures were

approved by the Animal Care Committee at the University of Toronto, in accordance

with the Guidelines for Canadian Council on Animal Care. Three weeks after the

initial ovariectomy animals were subjected to experimentation.

3.2 Stimulation Protocol All procedures were performed under anesthesia (isoflurane/oxygen gas

mixture; 1L/min). Once anesthetized, animals were oriented in a supine position on

a small-animal warming platform (806D, Aurora Scientific Inc., Aurora, Canada)

maintained at 37°C. The left knee was secured between two vertical stabilizing posts

by the insertion of a 25G x 1.5-inch needle through the hind limb in a lateral

orientation, directly distal to the condyles. Two 25G x 0.5-inch needle probe

electrodes (Chalgren Enterprises, CA, USA) were inserted subcutaneously in a

longitudinal position, directly along the TA muscle. Adhesive tape was applied to

secure the left leg to the pedal of a computer-controlled servomotor (301C, Aurora

23

Scientific Inc., Aurora, Canada). The pedal was adjusted three-dimensionally until

the secured ankle was in line with the knee and the knee angle at 120°.

Electrical stimulation was produced from a bi-phase stimulator (701C Aurora

Scientific Inc., Aurora, Canada), controlled by a computer hardware interface (604A,

Aurora Scientific Inc., Aurora, Canada) synchronized to the servomotor (Cambridge

Technology, model #6650LR) movements. Once the correct orientation was

established, optimal stimulation voltage was determined by stimulating the TA

muscle to cause dorsiflexion of the secured foot against a rigid pedal for 0.5-second

periods in intervals of 1V. Generally voltages between 8-12V stimulations were

provided. Isometric torque (g/cm) output was measured and the optimal

stimulation voltage for animals was selected based on the lowest voltage needed to

achieve peak torque.

Lengthening contraction stimulation was performed using a bi-phase

stimulator connected to a computer controlled hardware interface (604A, Aurora

Scientific Inc., Aurora, Canada) that could be adjusted using Dynamic Muscle Control

software (DMC; 610A, Aurora Scientific Inc., Aurora, Canada). Pre-treatment tetanic

tetanus values were used from the peak isometric torque data collected from single

optimized stimulation. Pre-twitch tension values were also determined prior to the

start of LCs.

Electrical stimulation was initiated for 0.2s during the treatment protocol

prior to any servomotor movement allowing for adequate muscle force

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

The servomotor pedal was initiated causing plantar flexion (LC) while remaining

24

contracted over a range of 38° for the next 0.3 seconds, causing an angular velocity

of 127°/s. Upon completion of each contraction, stimulation was stopped and the

pedal passively maneuvered the foot back to the respective start position within 3

seconds. LCs protocols consisted of 20 or 40 stimulated contractions, sectioned into

1 set of 20 LCs, with 3 seconds of rest between each repetition, or 2 sets of 20 LCs,

with 3 seconds of rest between each contraction and 5 minutes rest was allowed

between each set. After completion of the LCs, peak tetanic tension and twitch

tension were re-measured immediately after and at 10-minutes following LCS using

a single 0.5-seconds stimulation against a rigid pedal. Rats were monitored for one

hour following electrical stimulation while they recovered before being returned to

their cage.

3.3 Tissue Collection and Uterus Dissection Animals were euthanized 24 hours following LCs while under anesthesia via

cardiac exsanguination. Blood was collected by inserting an 18G needle into the left

ventricle and blood slowly removed, and placed in evacuated collection tubes

(Vacutainer, BD, NJ, USA) free of anti-coagulant agent heparin. Blood was mixed at

4° for two hours and centrifuged at 3,000 rpm for 20 minutes at 4°. Serum was

stored in 1.5ml eppendorf tubes in a -70°C freezer until later analysis (Appendix

7.1).

TA muscles were excised from all CC animals, OVI and OVX lengthening

contracted and contra-lateral (control), weighed and portioned into three pieces.

Muscle samples were either placed in 1.5ml eppendorf tubes and snap-frozen in

liquid nitrogen, or coated in optimal cutting temperature medium (OCT) mounted

25

onto a corkboard and rapidly frozen in isopentane immersed in liquid nitrogen.

Embedded muscles were stored at -70°C for later cryosectioning and morphological

analysis.

To determine if ovariectomy altered uteri mass, the uteri were removed after

making an incision along the midline of the body to access the uteri. Once removed,

excess fat was trimmed away, and if present, ovaries were removed (OVI and CC).

For consistency with each dissection the uterus was cut at the bottom before the

junction of the bicornate uterine horns. Excess fat and connective tissue was

removed prior to weighing (Appendix 7.2).

3.4 Biochemical Analysis 3.4.1 Tissue Homogenization Frozen sections of TA muscle (30-140mg) were homogenized in 10 volumes

of RIPA buffer (Sigma Aldrich, Cat #: R0278; 50 mM Tris-HCl, pH 8.0, with 150 mM

sodium chloride, 1.0% Igepal CA-630 [NP-40], 0.5% sodium deoxycholate and 0.1%

sodium dodecyl sulphate) with 1:100 volumes of protease inhibitor cocktail (Sigma

Aldrich, Cat #: P8340; 104mM of AEBSF, 0.085mM of Aprotinin, 4mM of Besatin,

1.4mM of E-64, 2mM of Leupeptin and 1.5mM of Pepstain A) per volume of RIPA

buffer. A Bead Bug Microtube Homogenizer (Model D1030, Product Code:

1143051039) was used to homogenize muscle samples by vibrating tissue three

times for 45 seconds each. Samples were placed on ice between homogenizations to

avoid an increase in temperature. Homogenized muscle was centrifuged

(Eppendorf, Model #: 5415 C) for 30 seconds at 14,000 rpm to remove connective

26

tissue and supernatants were aliquoted and stored in a -70° freezer for later

analysis (Appendix 7.3).

3.4.2 Muscle Protein Determinations

TA muscle protein concentrations were determined by the method of Lowry

et al. (1951) using bovine serum albumin (BSA) as a standard. In short, triplicate

tubes with 5μl of sample added to 0.5ml of ddH2O in a 13 x 100mm glass test tubes

were used. Five milliliters of Lowry reagent (5 ml/250 ml of 2% w/v CuSO45H₂O, 5

ml/250 ml 4% w/v sodium tartrate in 240 ml of 3% w/v Na2CO3) was added,

vortexed and allowed to react for a minimum of 10 minutes. Phenol reagent (0.5 ml;

Sigma Aldrich, Cat #: F9252) diluted 1:2 times with ddH₂O was added while

vortexing and allowed to react for 30 minutes. Absorbance was measured at 660

nm in a spectrophotometer (Turner, model # 340). A standard curve consisting of

20, 40, 60, 80 and 100 µg of BSA was constructed and were determined using a

linear regression equation (y=mx+b) and compared to the net absorbance of

samples (Appendix 7.4).

3.4.3 Protein Content Calculation

To calculate muscle protein content, the weight of the tissue sample (mg)

was added to the volume of homogenizing buffer and a total volume (ml) obtained.

The volume was multiplied by the determined protein concentration (Lowry from

3.52) to provide a total protein amount (g). The wet weight mass of the

homogenized muscle protein (mg) was divided by the muscle protein concentration

27

value obtained, yielding a protein-to-mass ratio (mg of protein/g wet weight

muscle). Total protein content of the TA muscle was expressed as a ratio of the LC to

the CL.

3.4.4 One-Dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

Known amounts (100μg -200μg) of muscle homogenates were separated by

one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE) as previously described (Laemmli, 1970), with the exception of the resolving

gel, prepared as a 5-15% linear gradient. Glass plates were cleaned with 70%

ethanol and assembled as described by the manufacture (Bio-Rad, Mississauga,

Canada). The separating gel consisted of two parts; a 5% and 15% acrylamide

mixture. The 15% acrylamide was added to the back chamber and stirring applied.

The stopcock was closed and the air between the two chambers removed. The 5%

acrylamide mixture was added to the gradient maker chamber away from the spout.

Once the 15% acrylamide covered bottom portion of the cast, the stopcock was

opened and the acrylamide solutions mixed and drained into the gel. A 4:1 mixture

of H₂O and butanol was gently overlaid to remove any bubbles and gels were left to

polymerize for 1 hour or overnight. A stacking gel consisting ddH2O, stacking buffer

(0.8% SDS, 12.1% Tris pH6.8) and 3% acrylamide was overlaid over the resolving

gel and a 10 well 1.5mm teflon comb placed in the gel solution and allowed to

polymerize for two hours. Muscle homogenates (100μg-200μg) were loaded into

wells and electrophoresed at 60 volts until the dye front entered the separating gel.

Once into the separating gel voltage was increased to 110 volts until the dye reached

28

the bottom of the gel. A Bio-Rad Power Pac 1000 was used (Bio-Rad, Mississauga,

Canada) (Appendix 7.5).

3.4.5 Western Blotting—Protein Analysis

SDS-PAGE separated proteins were transferred from gels to nitrocellulose

membranes (0.22μm pore size), (Bio-Rad Laboratories, Mississauga, Canada) using

the method of Towbin et al. (1979) and modified to the Bio-Rad mini-protein II gel

transfer system described by Frier et al. (2008). Following transfer (~2 hours),

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

powder (NFSM) or 3% BSA dissolved in Tris-buffered saline (TBS) for one hour at

room temperature. TBS plus Tween-20 (TTBS) was used to wash the membranes

twice for five minutes each before being incubated overnight at 4°C with specific

antibodies for Hsp25 (ADI-SPP-715, ENZO, USA), Hsp72 (ADI-SPA-812, ENZO, USA),

HSF1 (H-311, SC-9144, Santa Cruz), diluted in TTBS with 2% NFSM or 3% BSA in

1:1000 concentration. Membranes were washed twice in TTBS for 5 minutes and

incubated for one hour at room temperature with appropriate secondary Goat Anti-

rabbit IgG HRP-linked antibody (BioRad Cat# 170-6515) and diluted in TTBS plus

2% NFSM or 3% BSA in 1:1000 concentration. Membranes were washed twice in

TTBS for five minutes and once in TBS for 30 minutes before being developed using

C-Digit (Mandel Scientific, Model #: CDG-001073 3600). Nitrocellulose membranes

were saturated with appropriate readymade substrates of eight mL of Luminata

Forte Western HRP Substrate (Millipore Corporation, Billerica, MA, Cat #:

WBLUF0100). Each blot was incubated separately for two minutes and developed

29

using Li-Cor C-Digit IS version 3.1 (Mandel Scientific, Model #: CDG-001073 3600).

Membranes were placed face down on the measuring surface of the C-Digit and

scanned at six minutes at normal sensitivity (Hsp25 and 72) or twelve minutes at

high sensitivity (HSF1). The optimal density of bands was quantified using the

Image Studio software (Image Studio Digits, version 5.2.5) and expressed as a fold-

increase of lengthening contracted TA muscle divided by contra-lateral (control) TA

muscle and values were expressed relative to CC animals, which was set to a value of

one (Appendix 7.5).

3.4.6 ELISA—Blood Serum Analysis Systemic circulation of serum 17-β estradiol concentration was measured

using an enzyme-linked immunosorbent assay (ELISA) (Abcam, ab108667). Serum

estradiol samples were determined by comparing a ratio of kit 17-β estradiol

control to animal serum samples. Blood serum samples were compared to a

reference standard. ELISA kit was brought to room temperature while tissue

samples thawed. Once thawed, 25μl of standards, kit control and tissue samples

were added to appropriate wells. Following this, 200μl of 17β-estradiol HRP

conjugate was added to each well and the 96 well plate was covered with aluminum

foil and left to incubate for two hours at 37°. Following incubation, the well contents

were removed and washed with the three times with 300μl of wash solution and

any excess solution was blotted from the wells. 100μl of TBM substrate was added

to each well and allowed to incubate for precisely 30 minutes at room temperature.

Stop solution (100μl) was added to each well and the 96 well plate was read at a

wavelength of 450nm within 30 minutes of final incubation (Appendix 7.6).

30

3.5 Morphological Analysis 3.5.1 Muscle Cross-Sectioning

Portions of skeletal muscle preserved in OCT medium, were cut using a

cryostat at -20°C (Cryo-Cut, American Optical Company, model #: 830) at a

thickness of 10 microns. The sections were air dried and stored at -20°C freezer

until stained for visualization using hematoxylin and eosin (H&E) (Appendix 7.7).

3.6.2 Hematoxylin and Eosin Staining

Cross-sections were stained using H&E. Staining was performed by placing

the slides in tap water for one minute, followed by hematoxylin (Sigma Aldrich; Cat

#: HHS32) for 3 minutes, the slides were rinsed in fresh tap water and placed into

distilled water for one minute, respectively. Cross-sections were moved through a

series of ethanol solution (30% and 50%) for one minute, before being placed in

eosin (Sigma Aldrich; Cat #: HT110180) stain for seven minutes. Slides were then

rinsed in a series of ethanol solutions of 70%, 90% and 95% for one minute each.

Cross-sections were placed in the first 100% ethanol for two minutes and a second

100% ethanol solution for two minutes, before being moved to xylene substitute

(Sigma Aldrich; Cat #: A5597) five minutes. Cross-sections were sealed with a VWR

micro glass cover (Cat #: 48366 067) and sealed using cytoseal 280 glue (Thermo

Scientific; Cat #: 8311-4). A small drop of the cytoseal glue was placed in the center

of the micro glass cover and placed directly over the centre of the stained slide.

Direct pressure was applied over the glass cover to force out air bubbles. Once the

sealed slides fully dried, slides were viewed using a Zeiss Axioskop and

31

photographed at 5x, 10x and 40x magnification using a Canon EOS 70D (Appendix

7.8).

3.5.3 ImageJ Quantification

ImageJ 64-bit software (National Institutes of Health; version 1.48v) was

used to quantify characteristics of skeletal muscle damage following H&E staining.

One hundred muscle fibres of each sample were counted to get an accurate

representation of muscle damage in each sample group. Characteristics of muscle

damage included fibre area, circularity and roundness. In addition, following the

model by Rizo-Roca et al. (2015) necrotic fibres were examined, as well as

endomysial and perimysial infiltration and distension. To do this, photos of

micrometers for 5x, 10x and 40x magnification were used to set the scale to count

measure fibre area, circularity and roundness. Photos of the micrograph were

loaded into ImageJ and line tool was selected to draw a straight line across a known

distance micron distance. The distance in pixels measured was set to the known

distance in microns and globally set to apply to images of the same magnification.

To examine the extent of muscle fibre damage, TA muscle fibre values from

the lengthening contracted TA muscles were compared to the CL values to

determine if LCs resulted in increased skeletal muscle damage, or in TA muscles

between OVI and OVX animals to examine if ovariectomy had an effect on muscle

fibre changes. In addition, values from the CL TA muscle were also compared

between OVI and OVX animals. Two hundred muscle fibres were counted in each

32

sample group to get an indication of focal or global damage and differences between

OVI and OVX animals (Appendix 7.9).

3.5.4 Histological Quantification Fibre roundness, circularity and area were quantified using the ImageJ tools

using ImageJ software 64-bit for Mac OS X. To count fibre roundness, circularity and

area, the polygon tool was used to carefully trace around the fibre. Once a fibre was

measured the paintbrush tool was selected and an “X” was drawn through the fibre

to ensure the fibre was not counted a second time.

Fibre area was quantified by the selected area, measured in square pixels and

calibrated in square units (e.g. mm2, μm2). Circularity is a measure that ranges from

0 (infinite polygon) to 1 (perfect circle). In contrast, roundness better captures

asymmetrical shapes, thus overall shape is accounted for.

In addition, following methods published by Rizo-Roca et al. (2015) fibre

damage was examined through multiple measures including; necrotic fibres

characterized by the infiltration of inflammatory cells (myophagocytosis),

fragmented sarcoplasm, dark staining (hypercontracted) and pale staining (necrotic

fibres). Furthermore, the extracellular inflammatory domain was examined,

consisting of endomysium and perimysial infiltration. Permysial infiltration

consisted of small, mononuclear cells found in the perimysium, while endomyisal

infiltration was similar but found within the endomysium. Lastly, the interstitial

compartment was analyzed and divided into endomysial and perimysial distention,

where space (distension) was present between the individual muscle fibres or

33

fascicles, respectively. Fibres were counted and marked using the paintbrush tool to

avoid recounting fibres. Two hundred fibres from each muscle sample were counted

to get an accurate representation of muscle damage (Appendix 7.9).

3.6 Statistical Analysis For all measures means and standard error of the mean were calculated to

compare differences from CC, OVI and OVX animals. When data was merged

between CC and OVI (CC/OVI) animals (body mass, contra-lateral control TA mass,

uterus mass and 17-β estradiol concentration) a Students T-test was performed. For

analysis of fibre morphology and biochemical analysis a one-way ANOVA test with a

Tukey’s post hoc analysis was used. A two-way repeated measures ANOVA was used

to analyze all measures of TA muscle torque, maximal tetanic tension and maximal

tetanic twitch. Statistical significance at an accepted P value of 0.05 was set and

GraphPad Prism version 6 software was used to perform data analysis.

34

Chapter IV: Results

4.1 Anatomical and Physiological Alterations 4.1.1 Anatomical Changes Following Ovariectomy To determine if ovariectomy (estrogen-removal) resulted in anatomical

changes, the mass of the body, TA muscles and uteri were measured. Given that

animals from CC groups are biologically identical to OVI animals, except for the

stimulation provided to the TA muscles, in cases where no differences between

these groups (OVI and CC animals), were observed, data were combined. Thus,

when the body mass of OVI/CC animals (285.0 ±6.1g) was compared to OVX animals

(335.9 ±6.0g), a 17.9% increase in the latter was observed (P<0.001; Table 1),

suggesting one effect of ovariectomy is an increased body mass.

Given that ovariectomy resulted in an increased body mass, it was

determined if muscle mass also increased. To do this, mass from the CL TA muscle

from OVI/CC animals (0.56 ±0.01g) were compared to the mass of the CL TA

muscles from OVX animals (0.66 ±0.02g). A significant increase (P<0.001; Table 1)

in TA muscle mass was observed in the CL TA muscles from OVX animals. To

determine if the observed elevation in TA mass was related to the increase in body

mass or to muscle hypertrophy, muscle mass to body mass ratios were determined.

Since no differences were observed in the TA muscle mass to body mass ratios

between OVI/CC and OVX animals (1.99 ±0.04 vs. 1.96 ±0.03) (Table 1), the

increased TA mass in ovariectomized animals was the result of increased body

mass. That is, TA mass was proportional to body mass.

35

The uterus is known to be an estrogen responsive organ (Astwood, 1939;

Jazbutyte et al., 2006). Thus, three weeks of estrogen deprivation via ovariectomy

would be expected to alter uteri mass. To assess this, uteri were removed from OVX

animals and their mass compared to OVI/CC uteri (0.57 ±0.03g vs. 0.17 ±0.01g). A

marked decrease of 70% was observed in the uteri mass from OVX animals

(P<0.001; Table 1), suggesting the loss of ovaries/estrogen dramatically affected the

mass of this organ.

To confirm that ovariectomy decreased blood estrogen levels, serum 17-β

estradiol levels were measured by enzyme-linked immunosorbent assay. OVX

animals displayed a significantly lower serum estradiol concentration (-48.6%)

when compared to all OVI/CC animals (19.6 ±2.1pg/ml vs. 38.1 ±4.4pg/ml; P<0.01;

Table 1). Taken together, the atrophy of the uteri in combination with decreased

serum 17-β estradiol concentrations suggest both tibilais anterior muscle and

estrogen sensitive tissues were markedly altered by ovariectomy.

4.1.2 The Effect of Lengthening Contractions on TA Muscle Mass

To determine if LCs altered the mass of TA muscles from OVX animals, TA

mass was measured twenty-four hours following LCs. An increase in TA mass was

observed following 20 LCs; such that, the mass of the lengthening contracted TA

increased in OVX animals by 13.9%, compared to TA masses from OVI animals

(0.72±0.02 vs. 0.62 ±0.03g; P<0.05). Similarly, following 40 LCs, the TA muscles

from OVX animals showed a 12.7% increase in mass when compared to lengthening

contracted TA muscle from OVI animals (0.63 ±0.01 vs. 0.55 ±0.01g; P<0.05; Table

36

2). When TA mass was expressed relative to body mass significance was negated

suggesting the observed increase in TA mass is due to the increased body mass

following ovariectomy.

Given that muscle swelling may occur following muscle contractions

(McCully and Faulkner, 1985), the absolute (g) TA muscle mass from CL and LC

limbs were compared. No statistical significances were found in the TA masses

between OVI and OVX groups following either 20 or 40 LCs (Table 2). Thus, the LCs

provided (20 or 40) did not result in an increased in TA mass.

37

Table 1: The Effects of Ovariectomy on Body, Muscle and Uteri Masses and 17-β Estradiol Concentration

Ovary-intact (OVI/CC)

n=17

Ovariectomized (OVX) n=12

Percent Difference

Body mass (g) 285.0 ±6.1 335.9g ±6.0*** + 17.9% Contra-lateral Control Tibialis Anterior Mass (g)

0.55 ±0.01 0.66 ±0.02*** + 20.0%

TA Muscle Mass/Body Mass

1.99 ±0.04 1.96 ±0.03 -1.51%

Uterus mass (g)

0.57 ±0.03 0.17 ±0.01*** - 70.2%

17-β Estradiol Concentration (pg/ml)

38.1 ±4.4 19.6 ±2.1** - 48.6%

Animal masses were measured following ovariectomy and/or LCs, OVX animals displayed significantly greater body. Twenty-four hours following LCs, TA muscles and uteri were removed and measured. Uteri mass was significantly decreased in OVX animals. In addition, blood serum 17-β estradiol concentration was measured via an ELISA and decreased serum 17-β estradiol concentration in OVX animals was observed. Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend **= P<0.01, ***= P<0.001.

38

Table 2: Tibilais Anterior Muscle Mass in the Lengthening Contracted and Contra-lateral (Control) Limbs

OVI OVX

Control

Contra-lateral (control)

20 LC

Contra-lateral (control)

40 LC

Contra-lateral (control)

20 LC Contra-lateral (control)

40 LC

Tibialis Anterior Muscle (g)

0.60 ±0.02

0.58 ±0.03

0.62 ±0.03

0.51 ±0.02

0.55 ±0.01

0.69 ±0.03

0.72 ±0.02**

0.63 ±0.01

0.63 ±0.01*

Treatment TA/Contra-lateral Control TA

1.00

1.07 1.07 1.04 1.00

Characterization of the LC and CL TA mass and the ratio of LC limb divided by the CL, 24 hours following LC. Groups consisted of an n=5 for control TA muscles and an n=6 for both 20 and 40 LCs in each animal group. Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend *= P<0.05, **= P<0.01.

39

4.1.3 Muscle Protein Concentration

Protein concentration expressed as the amount of protein per mg muscle wet

weight was used to determine if the observed increase in OVX TA mass following

LCs was the result of increased protein concentration, an increased overall size or

merely due to water retention. For TA muscles subjected to LCs, a ratio of

lengthening contracted muscle mass value over CL mass was determined. For

animals not subjected (CC) to any LCs, a ratio of the right to the left TA muscle was

used. Ratios from the lengthening contracted limbs were compared to ratios of CL

TA muscle from CC animals. As expected, left and right TA muscle from CC animals

showed no differences in TA muscle protein content (ratio = 0.98 ±0.06). Following

20 LCs (1.0 ±0.08) or 40 LCs (1.0 ±0.08) the TA muscle protein content from OVI

animals was lower than CC animals although not statistically significant. Similarly,

no differences in TA muscle protein content from TA muscles from OVX animals was

detected when compared to CC animals. Thus, despite changes in the quantity of

muscle mass, muscle protein concentration was not significantly altered in the TA

muscle following ovariectomy.

4.2 Muscle Contractile Alterations 4.2.1 Muscle Contractile Properties To determine the effect of ovariectomy on skeletal muscle contractile

properties, TA muscle torque was measured throughout the 20 or 40 LCs as

described in methods and materials. Prior to undergoing LCs, maximal muscle

tetanic tension (MTT) and maximal twitch tension (MTw) were determined.

Following the cessation of LCs, tetanic tension and twitch tension were also

40

measured immediately after and at ten minutes following the last (20th or 40th) LC.

During an LC muscle torque can be separated into an active and a passive

component. The active component of torque is equal to MTT and was reached at

0.2ms, prior to lever movement (Figure 2). The passive component is the torque

resulting from the stretching of the TA muscle and occurred while the lever was

actively moving. The passive component of torque is an indirect measure of stretch

(or connective tissue tolerance) (Figure 1). To normalize for alteration in muscle

mass, such as that observed in OVX animals, data are expressed in relation to muscle

mass, known as specific torque. That is, torque (g/cm) divided by TA mass (mg) to

provide a value in g-cm/mg muscle wet weight.

Figure 1: The Active and Passive Components of TA Muscle Torque During a Lengthening Contraction A lengthening contraction can be divided into the active (A) and passive (B) components of a cotraction. The active component of torque is the summation of tetanic tension, and occurs prior to the lever moving. The passive component if torque develops after the lever movesm and is an indirect measure of connective tissue tolerance or stretch.

41

4.2.2 Specific Total Torque Throughout 20 and 40 Lengthening Contractions When compared to the first LC, TA muscle torque was progressively

decreased by the ninth contraction for both groups (OVI: 0.71 ±0.04 vs. OVX: 0.70

±0.04g-cm/mg; P<0.001; Figure 2). When specific torque was measured no

difference in TA muscle torque was observed between OVI and OVX animals at any

contraction throughout the 20 LCs.

In TA muscles subjected to 40 LCs, TA torque was gradually decreased

(P<0.001) in OVI animals such that, after the thirteenth contraction in the first set

(0.63 ±0.04g-cm/mg; compared to contraction one) torque decrease was significant.

In the second set, when compared to the first contraction, a significant decrease

(P<0.001) in TA muscle from OVI animals was detected after the fourth contraction.

When TA muscles from OVX animals were examined, a decreased (P<0.001) in TA

muscle torque (0.65 ±0.04g-cm/mg) was noted after the tenth contraction in the

first set. When compared to the first contraction in the second set, a significant

decrease (P<0.001; Figure 3) in TA muscles from OVX animals was detected by the

fifth contraction. Overall, given that the TA muscle torque of OVI and OVX animals

decreased at similar points, it suggests ovariectomy does not change the ability of

skeletal muscle to generate torque.

42

Figure 2: Specific Muscle Torque is Similar Between OVI and OVX Animals Throughout 20 Lengthening Contractions No differences in TA muscle torque between OVI and OVX animals were observed throughout 20 LCs. TA muscle torque decreased in the ninth contraction in both OVI and OVX animals (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend ***= P<0.001.

43

Figure 3: Specific Muscle Torque is Similar Between OVI and OVX Animals Throughout 40 Lengthening Contractions No differences in TA muscle torque were observed throughout 40 LCs between OVI and OVX animals. TA muscles from OVI animals decreased torque in the thirteenth and fourth contraction in set one and two, respectively (P<0.001). While TA muscles from OVX animals decreased torque in the tenth and fifth contraction in set one and two, respectively (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend ***= P<0.001.

44

4.2.3 The Active Component of Tibialis Anterior Muscle Torque The active component of muscle torque is a measure of tetanic tension prior

to the foot pedal actively lengthening the TA muscle and peaks between 0-0.2ms

following stimulation. To determine if the active component of TA muscle torque

was altered between OVI and OVX animals, the active component was measured and

compared for each LC. In TA muscles from OVI animals subjected to 20 LCs, the

active component of TA muscle torque was significantly decreased by the fifth

contraction, while TA muscles from OVX animals showed a significant decrease by

the seventh contraction (0.61 ±0.03 vs. 0.64 ±0.03g-cm/mg; P<0.001). No

differences were observed between the active components of TA muscle torque of

OVI and OVX animals for any of the repetition time points examined during the 20

LCs (Figure 4). Thus, ovariectomy does not appear to alter the active component of

muscle torque during lengthening contractions.

To investigate muscle torque beyond 20 LCs, TA muscles were subjected to

40 LCs. In this case, the active component of TA muscle torque from OVI animals

was significantly decreased (from the first contraction of the first set) at the eighth

contraction (0.54 ±0.04g-cm/mg; P<0.001). In the second set, TA muscles from OVI

animals showed a decreased (P<0.001) torque at the fifth contraction when

compared to the first contraction. Similarly, TA muscle torque from OVX animals

significantly decreased at the seventh contraction of the first set (0.58 ±0.04g-

cm/mg; P<0.001) and at the sixth contraction of the second set (compared to the

first contraction of the second set; P<0.001). No differences in the active component

of TA muscle torque were observed between TA muscles from OVI and OVX animals

45

at any repetitions during the 40 LCs. A similar pattern of active component TA

torque loss was observed between OVI and OVX animals during 40 LCs (Figure 5).

Taken together, these data suggest that although LCs tended to decrease in the

active component of TA muscle torque, ovariectomy does not result in any

additional losses in the active component of muscle torque.

46

Figure 4: Ovariectomy Does Not Alter the Active Component of Muscle Torque Throughout 20 Lengthening Contractions No differences were observed in the active component of TA muscle torque between OVI and OVX animals throughout 20 LCs. TA muscle from OVI animals decreased the active component of TA muscle torque at the fifth contraction from the first contraction (P<0.001). In contrast, TA muscle from OVX animals decreased the active component of TA muscle torque at the seventh contraction from the first contraction (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend ***= P<0.001.

47

Figure 5: Ovariectomy Does Not Alter the Active Component of Muscle Torque Throughout 40 Lengthening Contractions No differences were observed in the active component of TA muscle torque between OVI and OVX animals throughout 40 LCs. In TA muscle from OVI animals the active component of torque decreased at the eighth contraction compared to the first contraction (set one) (P<0.001) and fifth contraction compared to the first contraction in set two (P<0.001). In TA muscle from the active component of TA muscle torque decreased by the seventh contraction compared to the first contraction (set one) (P<0.001) and sixth contraction compared to the first contraction in set two (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend ***= P<0.001.

48

4.2.4 The Passive Component of Tibialis Anterior Muscle Torque The passive component of torque refers to the torque generated after MTT

has plateaued. This reflects the stretch of the muscle generated by the tension (pull)

from the foot pedal moving and occurs between 0.3ms to 1.0 seconds. The passive

component of torque may be considered as an indirect measure of muscle and

connective tissue resistance to stretch. For the ease of presentation, decreases in the

passive component of TA muscle torque are presented in relation to the peak TA

obtained during contractions (Figure 6 and 7). Following 20 LCs, this occurred at

repetition five for TA muscles from OVI animals and at repetition eight for TA

muscles from OVX animals. A steady decrease (P<0.001) in the passive component

of TA muscle torque occurred at the eleventh contraction in TA muscles from OVI

animals (0.10 ±0.01g-cm/mg). In contrast, the passive component of TA muscle

torque from OVX animals was gradually but significantly decreased (P<0.01) by the

sixteenth (0.07 ±0.01g-cm/mg). Again no differences were observed in the passive

component of TA muscle torque between OVI and OVX animals. However, it should

be noted that TA muscles from OVI animals reached their peak passive torque prior

to the TA muscles from OVX animals (fifth vs. eighth contraction; Figure 6). Taken

together, these data suggest that following ovariectomy, the passive component of

torque and perhaps connective tissue may stiffen and be able to resist greater TA

stretch.

During 40 LCs, no significant differences were observed in TA muscles

between OVI and OVX animals at any contraction time point. Decreases in the

passive component of TA muscle torque following 40 LCs are presented in relation

49

to the peak contraction torque (eight vs. seven, OVI vs. OVX). TA muscles from OVI

animals were decreased by the sixteenth contraction while TA muscles from OVX

animals were decreased by the eighteenth contraction during the first set (0.09

±0.01 vs. 0.07 ±0.005g-cm/mg; P<0.001, P<0.05, respectively).

When compared to the first contraction in the second set, TA muscle torque

from OVI animals showed a decreased passive component by the seventh

contraction while TA muscles from OVX animals showed a decrease by the ninth

contraction (P<0.001; Figure 7). When TA muscles from OVI and OVX animals were

compared, a trend of maintaining the passive component of TA muscle torque was

observed in OVX animals. In addition, TA muscles from OVI animals reached peak

passive torque prior to TA muscles from OVX animals. Lastly, although no difference

in the passive component of TA muscle torque was observed between OVI and OVX

animals throughout 40 LCs, the TA muscles from OVI animals lost the passive

component of torque prior to that of TA muscles from OVX animals. Thus,

ovariectomy may influence the onset of the passive component of TA muscle torque

loss throughout LCs.

50

Figure 6: Ovariectomized Animals Maintained the Passive Component of Torque Throughout 20 Lengthening Contractions No differences were observed in the passive component of TA muscle torque between OVI and OVX animals. The passive component of TA muscle torque from OVI animals decreased in the eleventh contraction from the peak of contraction five (P<0.001). In contrast, the passive component of TA muscle torque decreased in the TA muscle of OVX animals in the sixteenth contraction using the eighth contraction as the peak (P<0.01). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend **= P<0.01, ***= P<0.001

51

Figure 7: Ovariectomized Animals Maintained the Passive Component of Torque Throughout 40 Lengthening Contractions No differences were observed in the passive component of TA muscle torque between OVI and OVX animals. In TA muscle from OVI animals the passive component of torque decreased in the sixteenth contraction, while TA muscle torque from OVX animals decreased by the eighteenth in the first set (P<0.001, P<0.05, respectively). Both OVI and OVX animals were compared to their respective peak torque in the passive component. In the second set, the passive component of TA muscle torque decreased in the seventh contraction in TA muscles from OVI animals (P<0.001) when compared to the first contraction in the second set. Similarly, in the second set the passive component of TA muscle torque from OVX animals decreased by the ninth contraction, when compared to the first contraction. Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend *= P<0.05, **= P<0.01, ***= P<0.001.

52

4.2.5 Maximal Tetanic Tension Prior to and Following 20 and 40 Lengthening Contractions

To determine if ovariectomy altered the maximal tetanic tension of TA

muscle, measurements of MTT were reported prior to (pre), directly after (t=0) and

at 10-minutes after the last LC. Prior to 20 LCs, maximal tetanic tension was 0.64

±0.05g-cm/mg and 0.66 ± 0.04g-cm/mg in TA muscles from OVI and OVX animals,

respectively. Thus, prior to any LCs there was no significant difference in pre-tetanic

tension in TA muscles between OVI and OVX animals. Directly following the 20th LC

(t=0; 0.45 ±0.02g-cm/mg), TA muscle MTT from OVI animals was decreased

(P<0.001) by ~31% when compared to pre-tetanic tension. Similarly, TA muscle

MTT of OVX animals directly after (t=0; 0.43 ±0.02g-cm/mg) the 20th LC was also

significantly (P<0.001) decreased (~ 26%) when compared to pre-tetanic tension.

After ten minutes of recovery following the 20th LC, TA MTT (0.49 ±0.03g-

cm/mg) remained significantly (P<0.01) decreased in TA muscles from OVX animals

by ~35% compared to pre-MTT. In contrast, MTT TA tension of OVI animals (0.55

±0.02g-cm/mg) recovered such that is was not significantly reduced at ten minutes

following the last LC when compared to pre-MTT. This suggets ovariectomy may

alter the ability of skeletal muscle to recover following moderate LCs. No differences

were observed in MTT in TA muscles between OVI and OVX animals at any of the

three time points in 20 LCs (Figure 8).

In animals subjected to 40 LCs, TA muscle MTT was 0.68 ±0.03g-cm/mg and

0.70 ± 0.04g-cm/mg for OVI and OVX animals, prior to any LCs and showed no

significant difference in TA muscle pre-MTT between OVI and OVX animals. Directly

after (t=0) the 40th LC, MTT of TA muscles from OVI and OVX animals was

53

significantly (P<0.001) decreased (~60 and 61%; 0.27 ±0.02 vs. 0.27 ±0.05g-

cm/mg) when compared to pre-tetanic tension. At 10 minutes after the 40th LC, TA

muscle tetanic tension remained significantly (P<0.001) decreased when compared

to pre-tetanic tension for both OVI and OVX (~37% vs. 34%) TA muscles (0.43

±0.02 vs. 0.46 ±0.05g-cm/mg). When compared to tetanic tension directly after

(t=0) the 40th LC, the TA tetanic tension had significantly (P<0.001) recovered in

TAs from both OVI and OVX animals to 63% and 65% of pre-tetanic tension values

(Figure 9). However, no differences were observed in TA muscle tetanic tension

between OVI and OVX animals at any of the three time points examined.

54

0.0

0.2

0.4

0.6

0.8

Time

Teta

nic

Ten

sio

n (

g-c

m/m

g m

uscle

wet

weig

ht)

OVI

OVX

*****

Pre 0 +10 Pre 0 +10

***

NS

NS

NS

Figure 8: Maximal Tetanic Tension is Similar Between TA Muscles from OVI and OVX Animals Following 20 Lengthening Contractions No differences were observed in TA muscle MTT between OVI and OVX animals. Immediately following the 20th LC, TA MTT was significantly reduced in TA muscle from both OVI and OVX animals (P<0.001). Ten minutes following 20th LC, MTT was significantly decreased in only OVX animals (P<0.01). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend **= P<0.01, ***= P<0.001.

55

0.0

0.2

0.4

0.6

0.8

Time

Teta

nic

Ten

sio

n (

g-c

m/m

g m

uscle

wet

weig

ht)

OVI

OVX

***

***

******

***

***

Pre 0 +10 Pre 0 +10

NS Figure 9: Maximal Tetanic Tension is Similar Between TA Muscles from OVI and OVX Animals Following 40 Lengthening Contractions No differences were observed in TA muscle MTT between OVI and OVX animals. Immediately after and ten minutes following the 40th LC TA muscle MTT from both OVI and OVX animals compared to pre-MTT (P<0.001). In addition, post 10-minute recovery MTT was increased in TA muscle from both OVI and OVX animals when compared to TA muscle MTT directly the 40th LC (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend***=P<0.001.

56

4.2.6 Maximal Twitch Tension Prior to and Following 20 and 40 Lengthening Contractions Maximal twitch tension (MTw) is defined as the force generated from a single

muscle contraction in response to a single stimulus and was used to measure muscle

recovery following LCs. Similar to MTT, MTw was measured prior to (pre), directly

after (t=0) and at 10 minutes after the last LC. Prior to any LCs, TA MTw was 0.09

±0.01g-cm/mg for OVI animals and 0.12 ±0.02g-cm/mg for OVX animals and was

not significantly different. Directly after (t=0) the 20th LC, TA muscle twitch tension

was not significantly decreased in OVI animals or OVX animals (OVI: 0.10 ±0.01;

OVX: 0.10 ±0.01g-cm/mg) when compared to pre-MTw. At 10 minutes after the last

LC, both OVI and OVX TA muscle MTw was significantly decreased (P<0.01;

P<0.001), respectively when compared to pre-MTw. Ten minutes after the 20th LC

MTw was significantly decreased (P<0.001) in both TA muscle from OVI and OVX

animals when compared to MTw directly after the 20th LC (Figure 10). When the TA

muscles from OVI and OVX animals were compared, no differences were observed

between treatments at any of the three contraction time points examined.

To determine if an additional set of LCs influenced muscle function, animals

were subjected to a second set of 20 LCs. Similar to 20 LCs, TA muscle twitch tension

was measured at identical time points and no differences in maximal TA muscle pre-

twitch tension were observed between OVI and OVX animals. When compared to

pre-twitch tension (0.11 ±0.01g-cm/mg) TA muscles from OVI animals were

significantly (P<0.001, all conditions) decreased after the 40th LC such that a ~64%

loss at 0- (0.04 ±0.01g-cm/mg) and ~82% reduction at 10 minutes (0.02 ±0.003g-

cm/mg) was observed. When pre-TA muscle twitch tension (0.11 ±0.01g-cm/mg)

57

from OVX animals was compared to 0- (0.04 ±0.01g-cm/mg) and 10 minutes (0.04

±0.01g-cm/mg) a significant (P<0.001) decrease of approximately 64% for both

time points was observed. Notably, when compared to pre-twitch tension, TA

muscles from OVX animals recovered 36% of TA pre-twitch tension, while TA

muscles from OVI animals only recovered 18% at 10-minutes after the 40th LC.

However, similar to 20 LCs, no significant differences in TA twitch tension between

OVI and OVX animals were observed at any of the three time points examined

(Figure 11).

58

0.00

0.05

0.10

0.15

Time

Tw

itc

h T

en

sio

n (g

-cm

/mg

mu

sc

le

we

t w

eig

ht)

OVI

OVX

**

***

***

***

Pre 0 +10 Pre 0 +10

NS Figure 10: Maximal Twitch Tension is Decreased Following 20 Lengthening Contractions No differences were observed in TA muscle MTw between OVI and OVX animals. No difference was observed in TA muscle pre-MTW when compared to TA muscle MTw directly following the 20th LC in both OVI and OVX animals. At ten minutes following the 40th LC TA muscle MTw was significantly decreased in 10- compared to pre-MTw from both OVI and OVX animals (P<0.01, P<0.001, respectively). Furthermore, post 10-minute MTw was significantly reduced in the TA muscles of both OVI and OVX animals when compared to MTw directly after the 20th LC (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend **= P<0.01, ***= P<0.001.

59

0.00

0.05

0.10

0.15

Time

Tw

itc

h T

en

sio

n (g

-cm

/mg

mu

sc

le

we

t w

eig

ht)

OVI

OVX

***

***

***

***

Pre 0 +10 Pre 0 +10

NS

NS NS

Figure 11: Maximal Twitch Tension is Decreased Following 40 Lengthening Contractions No differences were observed in TA muscle MTw between OVI and OVX animals. In the TA muscle from both OVI and OVX animals, MTw was significantly decreased at 0 and 10-minute following the 40th LC when compared to TA muscle pre-MTw (P<0.001). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend ***= P<0.001.

60

4.3 Muscle Fibre Morphological and Histological Changes Following Ovariectomy and/or Lengthening Contractions 4.3.1 Histological Measures of Muscle Damage To determine if ovariectomy and/or exposure to LCs resulted in differences

in muscle damage, TA muscle cross-sections from both the CL and lengthening

contracted muscles were stained with H&E and examined by light microscopy. Fibre

area, circularity and roundness were measured (Table 3). Photomicrographs were

quantified using ImageJ as described in methods and material. In addition, a scale of

muscle fibre damaged developed by Rizo-Roca et al. (2015) was used to examine the

extent of muscle fibre necrosis, endomysial and perimysial infiltration of immune

cells and distension. The scale used a score of 0 as the absence of damage, 1 as

moderate damage and 2 as severe damage [Rizo-Roca et al. (2015)] (Table 4).

4.3.2 Muscle Fibre Morphology in the Contra-lateral (Control) TA Muscles from OVI and OVX Animals

Fibre area of CL TA muscles from OVX animals were 17.5% greater (P<0.001)

when compared to TA muscle fibre area from OVI animals (4169 ±60.0 vs. 3441 ±

53.0μm2). The CL TA muscles from OVX animals also displayed a 7.3-fold increase

(P<0.01) in endomysial distension (altered space between the individual muscle

fibres), when compared to the CL from OVI animals. Necrotic fibres of CL TA

muscles from OVX animals were increased 7.5-fold compared to CL TA muscles from

OVI animals (OVI: 0.06 ±0.06 vs. OVX: 0.45 ±0.15).

61

4.3.3 Visual Analysis of Muscle Damage from TA Muscles from OVI and OVX Animals Subjected to Lengthening Contractions

Visible signs of muscle damage include fibre necrosis, rounded fibres and

endomysial infiltration of immune cell. Figures 12-14 show photomicrographs of TA

muscle fibres from 20 or 40 LCs from OVI and OVX animals stained by H&E and

visualized using light microscopy. Necrotic fibres were considered those with

infiltrating inflammatory cells, fragmented sarcoplasm and dark or lightly stained

fibres [Rizo-Roca et al. (2015)]. In addition, endomysial infiltration of immune cells

was characterized by the presence of mononuclear cells within the muscle fibres.

Figure 12, panels A and F show fibre necrosis following both 20 and 40 LCs. The

presence of rounded fibres appears to be increased following LCs (Figure 13; Panel

C, Figure 14; Panel A). An increased endomysial infiltration was also observed

following 20 and 40 LCs (Figure 14; Panel A and D). Thus, LCs resulted in the

appearance of an increased extent of muscle fibre damage markers in the TA

muscles from both OVI and OVX animals.

4.3.4 TA Muscle Fibre Area from OVI and OVX Animals Following Lengthening Contractions

When LC TA muscle fibre area from OVI animals were compared to OVI

animals, TA fibres area from OVX animals was increased (P<0.001) by 21%

following 20 LCs (OVX: 4010 ±83.2 vs. OVI: 3159 ±61.9μm2). When subjected to 40

LCs, TA muscles from OVI animals only showed a 9.7% increase (3810 ± 75.9μm2;

P<0.01) in fibre area when compared to TA muscles from the CL. These findings

suggest estrogen deprivation alters fibre area when subjected to LCs.

62

4.3.5 TA Muscle Fibre Circularity from OVI and OVX Animals Following Lengthening Contractions

To further confirm if ovariectomy altered fibre morphology, fibre circularity

was measured. When compared to the CL TA muscles, no differences in fibre

circularity was observed in lengthening contracted TA muscle from OVI animals

following 20 LCs. In contrast, when compared to the CL TA muscles, lengthening

contracted TA muscles from OVX animals showed a small but significant (3.6%)

increase (P<0.001) in fibre circularity following 20 LCs. Following 40 LCs, TA muscle

fibres from OVI animals also exhibited a small but significant (2.4%; P<0.001)

increase in the muscle fibre circularity measure when compared to their respective

contra-lateral (control) TA muscles. Similarly, when compared to the CL TA muscle,

lengthening contracted TA muscles from OVX animals also showed a 3.6% increase

(P<0.001) in fibre circularity when subjected to 40 LCs. No differences in fibre

circularity were observed in CL or LC TA muscles between OVI and OVX animals.

4.3.6 TA Muscle Fibre Roundness from OVI and OVX Animals Following Lengthening Contractions

Since muscle damage from LCs is known to cause fibre swelling (McCully and

Faulkner, 1985), muscle fibre roundness was also examined. Following 40 LCs, TA

muscle fibres from OVI animals displayed an increased measure of roundness

(4.0%; P<0.001) when compared to their respective CLs. Similar to OVI animals,

OVX animals also displayed an increased (5.0%; P<0.001) roundness measure

following 40 LCs when compared to their respective CLs.

63

Figure 13, panel A and figure 14, panel C show photomicrographs of rounded

in TA muscle fibres subjected to LCs from OVI and OVX animals, respectively. A

representative rounded TA muscle fibre from an OVI animal subjected to 20 LCs is

presented in figure 13, panel A while a fibre from an OVX animal following 40 LCs is

shown in figure 14, panel C. Perhaps not surprisingly, LCs resulted in increased fibre

roundness. Fibre roundness was greater in the lengthening contracted TA muscles

from both OVI and OVX animals subjected to 40 LCs when compared to fibres from

unstressed contra-lateral (control). When the CL TA muscles were compared to the

lengthening contracted TA muscles, no differences in TA muscle fibre roundness

between muscle fibres from OVI and OVX animals was observed.

4.3.7 TA Muscle Fibre Necrosis from OVI and OVX Animals Following Lengthening Contractions

The scale by developed by Rizo-Roca et al. (2015) considers necrotic fibres as

those with infiltrating inflammatory cells, fragmented sarcoplasm and dark or

lightly stained fibres. When compared to the CL TA muscles, TA muscles from OVI

animals subjected to 20 LCs showed a 18-fold increase (P<0.01) in necrotic fibre

score. Similarly a 17-fold increase (P<0.05) was observed in necrotic fibre score

following 40 LCs. In contrast, lengthening contracted TA muscles from OVX animals

subjected to 20 LC, showed only a 3.1-fold increase (P<0.01) in necrotic fibre score

when compared to their respective CL TA muscles, similarly a 3.3-fold increase

(P<0.01) in necrotic fibre score was observed following 40 LCs (Figure 14; Panel B).

The difference in fold increase between OVI and OVX was due to the decreased basal

necrotic fibres in OVI animals compared to OVX animals (Table 4). When TA

64

muscles from OVI and OVX animals subjected to 20 or 40 LCs were compared, no

differences in the extent/number of necrotic fibres were observed. Thus, an

increased presence of necrotic muscle fibres in TA muscle from OVI and OVX

animals was only observed following 20 or 40 LCs.

4.3.8 Endomysial Infiltration of TA Muscle Fibres from OVI and OVX Animals Following Lengthening Contractions The endoymisum encases individual muscle fibres and is the smallest

component of connective tissue of the muscle. To determine if this was altered

following ovariectormy and/or LCs, endomysial infiltration of immune cells was

measured as described in methods and materials. Muscle fibre (endomysial)

infiltration in OVI animals was un-altered following 20 LCs. However, following 40

LCs, infiltration was observed such that TA muscles showed a 3.6-fold increase

(P<0.01) in endomysial infiltration [score of mononuclear cells (endomysial

infiltration)] when compared to their respective CL TA muscles (Figure 14; Panel C).

A photomicrograph of TA muscle fibres from an OVI animal subjected to 40 LCs

displaying an increased endomysial infiltration is presented in Figure 12, panel G. In

contrast, TA muscles from OVX animals showed a dramatic 8.7-fold increase

(P<0.001) in the score of mononuclear cells (endomysial infiltration) following both

20 and 40 LCs when compared to their respective CL TA muscles (0.15 ±0.08).

Photomicrographs showing an increased immune cell infiltration in the TA muscle

fibres from OVX animal after 20 and 40 LCs are presented in Figure 12, panel F and

Figure 14, panels A and D, respectively. When TA muscle fibres were subjected to 20

65

or 40 LCs, no differences in endomysial infiltration of immune cells in TA muscle

fibres between OVI and OVX animals were detected.

4.3.9 Endomysial Distension of TA Muscle Fibres from OVI and OVX Animals Following Lengthening Contractions Endomysium distension is characterized by an altered or distended space

between individual muscle fibres. When CL TA muscles subjected to 20 LCs from

OVI animals were compared muscles, showed a 9.0- and 10.0-fold increase (P<0.01)

in the endomysial distension score following 20 and 40 LCs, respectively. In

contrast, TA muscle fibres from OVX animals subjected to LCs showed no increase in

endomysial distension following either 20 or 40 LCs.

4.3.10 Perimysial Infiltration and Distension of TA Muscle Fibres from OVI and OVX Animals Following Lengthening Contractions

The perimysium consists of the sheath of connective tissue surrounding a

bundle of muscle fibres. When perimysial infiltration (altered mononuclear cells in

the perimysium) and distension (altered space between the muscle fascicles) was

examined, no observable differences were detected between TA muscle fibres from

OVI and OVX animals, or between LC and CL TA muscle fibres following 20 or 40

LCs.

66

Figure 12: Skeletal Muscle Changes in Ovary-Intact and Ovariectomized Animals Following 20 or 40 Lengthening Contractions A visual representation showing skeletal muscle focal damage at 24 hours following 20 or 40 LCs. Panel A: OVI CL; Panel B: OVX CL; Panel C: OVI CL; Panel D: OVX CL. Panel E: 20 LC OVI, Panel F: 20 LC OVX, Panel G: 40 LC OVI and Panel H: 40 LC OVX fibres were stained using H&E as described in methods and materials and scale bars represent 100μm.

67

Figure 13: Skeletal Muscle Damage Following Lengthening Contractions in Ovary-intact Animals Focal damage at 24 hours following either 20 or 40 LCs in OVI animals. Panels A and B: muscle fibres following 20 LCs and Panel C and D: muscle fibres following 40 LCs. Panel A: displays a rounded fibre (denoted by the *). Panel B: is a fibre that has been surrounded and infiltrated by immune cells (denoted by the pointing arrow). Panel C: represents fibre infiltration by immune cells (signified by an X). Lastly, Panel D: is characterized by central nuclei (denoted by the pointing arrows). Fibres were stained using H&E as described in methods and materials. Scale bars represent 100µm.

68

Figure 14: Skeletal Muscle Damage in Ovarectomized Animals Following Lengthening Contractions Focal damage at 24 hours following either 20 or 40 LCs in OVX animals. Panels A and B: muscle fibres following 20 LCs and Panel C and D: muscle fibres following 40 LCs. Panel A: represents fibre infiltration from immune cells (denoted by the *). Panel B: displays fibre splitting and the transition to a ghost fibre, along with immune cell infiltration (signified by the #). Panel C: is a rounded fibre (denoted by the X). Lastly, Panel D: is characterized by immune cell infiltration of a rounded fibre (denoted by the diagonal arrow). Fibres were stained using H&E as described in methods and materials. Scale bars represent 100µm.

69

Table 3: Skeletal Muscle Fibre Alterations Following 20 or 40 Lengthening Contractions A B C D E F Fibre Histology

OVI CL (n=10)

OVX CL (n=10)

OVI 20 LC (n=5)

OVX 20 LC (n=5)

OVI 40 LC (n=5)

OVX 40 LC (n=5)

Area (μm2) 3441 ±53.0 4169 ±60.0A 3159 ±61.9 4010 ±83.2B/C 3810 ±75.9D 4000 103.1 Circularity 0.82 ±0.002 0.81 ±0.002 0.83 ±0.003 0.84 ±0.003B

0.84 ±0.003A

0.84 ±0.003B

Roundness 0.67 ±0.003 0.68 ±0.004 0.67 ±0.006 0.69 ±0.007 0.70 ±0.006A 0.72 ±0.006B Quantified measures of fibre area, circularity and roundness in unstressed CL TA and LC TA muscles from OVI and OVX animals. ASignificant in relation to OVI CL (P<0.001), BSignificant in relation to OVX CL (P<0.001), CSignificant in relation to 20 LC OVI (P<0.001), DSignificant in relation to 40 OVI (P<0.001). TA muscle fibre area was increased in the CL TA muscle from OVX animals, when compared to CL TA muscles from OVI animals (A). In addition, following 20 LCs, TA muscles from OVX animals showed increased fibre area when compared to the TA muscles from CL (B). OVI animals increased TA muscles fibre area after 40 LCs (D). Furthermore, following 20 LCs TA muscles from OVX animals increased fibre area when compared to OVI animals (C). TA muscles fibre circularity increased in OVI animals following 40 LCs (A) and in TA muscles from OVX animals (B). After 20 LCs, fibre circularity was increased in TA muscles from OVX animals (B). Lastly, roundness increased in TA muscles from both OVI and OVX animals following 40 LCs when compared to CL TA muscles (A and B). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM.

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Table 4: Skeletal Muscle Fibre Damage Following 20 or 40 Lengthening Contractions A B C D E F Histological Markers of Damage

OVI CL (n=10)

OVX CL (n=10)

OVI 20 LC (n=5)

OVX 20 LC (n=5)

OVI 40 LC (n=5)

OVX 40 LC (n=5)

Necrotic Fibres

0.06 ±0.06 0.45 ±0.15A 1.1 ±0.28A 1.4 ±0.22B

1.0 ±0.26C

1.5 ±0.27B

Endomysial Infiltration

0.39 ±0.14 0.15 ±0.08 0.60 ±0.27

1.3±0.3B

1.4 ±0.27A

1.3±0.26B

Endomysium Distension

0.11 ±0.08 0.8 ±0.09A 1.0 ±0.26A

1.1 ±0.28

1.1 ±0.23A

1.1 ±0.18

Presented are the quantified scores of necrotic fibres, endomysium infiltration and distension in the TA muscles of the CL and LC TA muscles. ASignificant in relation to OVI CL (P<0.01), BSignificant in relation to OVX CL (P<0.01), CSignificant in relation to OVI CL (P<0.05). Necrotic fibres increased in CL TA muscle fibres from OVX animals (A). Necrotic fibres were increased in the TA muscles from both OVI and OVX animals following both 20 (A and B) and 40 LCs (C and D). After 20 LCs, endomysial infiltration increased in TA muscles from OVX animals when compared to the CL TA muscles (B). In addition, endomyisal infiltration increased in the TA muscles from both OVI and OVX animals following 40 LCs when compared to the CL TA muscles (A and B). Lastly, endomysial distension increased in the CL TA muscles from OVX animals when compared to CL TA muscles from OVI animals (A). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM.

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4.4 The Cellular Stress Response Following Ovariectomy and/or Lengthening Contractions 4.4.1 The Constitutive Expression of Heat Shock Protein 25 in Tibialis Anterior Muscle

Hsps may be used as markers of stress following exercise, or they may reflect

the extent of cell protection prior to a stress. In this study, a three-week period of

ovariecotmy was provided rending the possibility such that muscle (fibre) changes

in gene expression may have occurred. To determine if the constitutive expression

of Hsp25 and/or 72 were altered in TA muscles from OVI and OVX animals, Hsps

were assessed by Western blotting in unstressed muscles (not subjected to LCs).

Following band quantification, a ratio of right over left limb was determined for CC

animals, while TA muscle exposed to LCs had the Hsp values expressed as LC over

CL. When expressed to CC animals (1.0 ±0.16), no difference was found between the

TA muscle Hsp25 content for OVI and OVX animals CL TA (Figure 15). This suggests

estrogen deprivation does not alter the constitutive expression of Hsp25.

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Figure 15: Constitutive Hsp25 Content is Unchanged Following Ovariectomy Characterization of constitutive expression of TA muscle Hsp25 from both OVI and OVX animals. No differences were observed in the CL TA muscle between OVX and OVI animals. Panel A: shows Western Blot bands for Hsp25. Lane 1: represents band intensity from the TA muscle from CC animals, Lane 2: depicts band intensity from the TA muscle from OVI animals and Lane 3: shows the band intensity from TA muscle from OVX animals. Panel B: displays the percent increase Hsp content (control-control right limb/contra-lateral control limb). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM.

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4.4.2 The Constitutive Expression of Heat Shock Protein 72 in Tibialis Anterior Muscle To determine if ovariectomy altered the constitutive level of Hsp72 TA

muscle content, Hsp72 content was measured in the unstressed CL TA muscles from

CC, OVI and OVI animals. When expressed to CC animals, no change in TA muscle

Hsp72 content from OVI animals was observed. However, Hsp72 content in TA

muscles from OVX animals showed a marked reduction such that Hsp72 content

was significantly (P<0.05) decreased by 57%, (0.57 ±0.08 vs. 0.99 ±0.14; Figure 16).

Thus, estrogen deprivation (ovariectomy) appears to significantly decrease the

constitutive expression of HSp72 content in skeletal muscles.

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Figure 16: Constitutive TA Muscle Hsp72 Expression is Significantly Decreased Following Ovariectomy Characterization of constitutive expression of TA muscle Hsp72 from both OVI and OVX animals. TA muscle from OVX animals displayed significantly less Hsp72 expression compared to TA muscle from OVI animals (P<0.05). Panel A: shows Western Blot bands for Hsp72 expression. Lane 1: shows band intensity of TA muscle from CC animals, Lane 2: represents band intensity of TA muscle from OVI animals, Lane 3: displays the band intensity of TA muscle from OVX animals. Panel B: displays the percent increase Hsp content (control-control right limb/contra-lateral control limb). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend *= P<0.05.

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4.4.3 TA Muscle Heat Shock Protein 25 Content Following Lengthening Contractions To examine the CSR in TA muscles from OVI and OVX animals, Hsp25 content

was examined 24 hours after LCs and the removal of the TA muscle. Given that the

constitutive expression of Hsp25 was similar between CC, CL OVI and CL OVX

animals, Hsp25 content was expressed as a ratio of the lengthening contracted value

relative to its respective CL value. In animals not subjected to LCs a ratio of right

over left TA muscle was computed. When compared to CC animals, no difference

was observed in TA muscle Hsp25 content from OVI animals following 20 LCs.

However, when subjected to 40 LCs TA muscle Hsp25 content from OVI animals

increased (P<0.01) by 260% (Figure 17). In comparison, TA muscle Hsp25 content

from OVX animals was increased (P<0.05) by 190% following 20 LCs and increased

(P<0.01) by 240% following 40 LCs (Figure 17). These data show that although

skeletal muscles from OVI and OVX animals are both capable of mounting a cellular

stress response, the stress may be experienced differently.

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Figure 17: TA Muscle Hsp25 Content Increases Following Lengthening Contractions TA muscle Hsp25 content was increased in the TA muscle from OVX animals 24 hours following 20 and 40 LCs (P<0.05, P<0.01, respectively) and in TA muscle from OVI animals following 40 LCs (P<0.01) compared to the TA muscle from CC animals. Panel A: shows Western bands for TA muscle Hsp25 content. Lane 1: shows band intensity of TA muscle from CC animals. Lane 2: displays band intensity of TA muscle from OVI animals following 20 LCs. Lane 3: depicts band intensity of TA muscle from OVX animals following 20 LCs and Lane 4: represents band intensity of TA muscle from OVI animals following 40 LCs. Lane 5: shows band intensity of TA muscle from OVX animals following 40 LCs. Panel B: the percent increase of TA muscle Hsp25 content (LC divided by CL). Data was displayed in one graph for clarity. Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Legend *= P<0.05, **= P<0.01.

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4.4.4 Heat Shock Protein 72 Content in the Lengthening Contracted TA Muscle

Given that Hsp72 content was decreased in the TA muscles from the CL OVX

animals, it was not possible to express a ratio of lengthening contracted TA value to

its respective CL value. To control for the decreased constitutive expression of

Hsp72 in OVX animals, Hsp72 content has been expressed relative to the value

obtained from CC animals. The Hsp72 content of TA muscles subjected to

lengthening contractions from OVI animals showed no increase following 20 LCs

when compared to CC animals (Figure 18). However, following 40 LCs, the TA

muscle Hsp72 content of OVI animals showed a 5-fold increase (P<0.05) when

compared to TA muscles from CC animals. In contrast, Hsp72 TA muscle content

from OVX animals remained unchanged following both 20 and 40 LCs when

compared to CC animals. These data suggest skeletal muscle from ovariectomized

animals demonstrates a compromised CSR in that its capacity to protect skeletal

muscle may be decreased due to decreased Hsp72 content following LCs.

78

Figure 18: TA Muscle Hsp72 Content Following 20 and 40 Lengthening Contractions in Ovary-Intact and Ovariectomized Animals TA muscle from both OVI and OVX animals did not increase Hsp72 content following 20 LCs. However, after 40 LCs, the TA muscle from OVI animals increased (P<0.05) compared to TA muscle from CC animals. Hsp72 content was not increased in TA muscle from OVX animals following LCs. Panel A: shows Western bands for TA muscle Hsp72 content. Lane 1: shows band intensity of TA muscle from CC animals. Lane 2: displays band intensity of TA muscle from OVI animals following 20 LCs. Lane 3: depicts band intensity of TA muscle from OVX animals following 20 LCs and Lane 4: represents band intensity of TA muscle from OVI animals following 40 LCs. Lane 5: shows band intensity of TA muscle from OVX animals following 40 LCs. Panel B: the percent increase of TA muscle Hsp72 content (LC divided by CL). Statistical significance was set at a P<0.05 and data are expressed as mean ±SEM. Data was displayed in one graph for clarity. Legend *= P<0.05.

79

4.4.5 HSF1 Content Stress induced increases in Hsps are regulated through the trimerization and

binding of heat shock factor-1 (HSF1) to the heat shock element (HSE) (Sarge et al.,

1993). The observed increase in TA muscle Hsp content following 20 or 40 LCs in

OVI and OVX animals suggests involvement of an HSF in the regulation of Hsp

expression following LCs. When HSF1 content was examined by Western blotting, a

ratio of CL TA muscles from OVI and OVX animals were compared to the value from

CC animals. A decrease in HSF1 content in the TA muscles of OVX animals was noted

when compared to TA muscles from OVI animals. Although a 31.0% reduction in

HSF1 content of TA muscle was observed in OVX animals, significance was not found

(Figure 19).

80

Figure 19: Heat Shock Factor1 Content in TA Muscles from OVX Animals No significant differences were observed in HSF1 content in TA muscle from OVI and OVX animals. Panel A: shows Western Blot bands for TA muscle HSF1 content and Panel B: displays the ratio of TA muscle HSF1 content between OVI and OVX animals. Lane 1: shows band intensity of TA muscle from OVI animals, Lane 2: shows band intensity of TA muscle from OVX animals. Data are expressed as mean ±SEM.

81

4.5 Summary 4.5.1 Anatomical, Physiological and Muscle Contractile Changes Following Ovariectomy and/or Lengthening Contractions Both body and TA mass were increased in OVX animals when compared to

OVI animals; however, uteri mass was markedly decreased in OVX animals. LCs

resulted in decreased torque throughout 20 and 40 LCs, as well as decreased tetanic

tension and twitch tension in all TA muscles examined. Ovariectomy did not

influence TA muscle MTT and MTw and no observable differences in TA muscles

torque were found throughout 20 or 40 LCs between OVI and OVX animals. The

passive component of LCs showed a decrease in TA muscle torque from OVI animals

prior to that observed from OVX animals, suggesting that an earlier onset of torque

loss in the passive component of torque may be mediated by estrogen. Lastly, the

active component of torque during LCs was also not affected by estrogen

deprivation.

4.5.2 The Changes in TA Muscle Fibre Morphology Following Ovariectomy and/or Lengthening Contractions

Ovariectomy resulted in an increased fibre area in the CL TA muscles from

OVX animals, when compared to CL TA muscles from OVI animals. CL TA muscles

from OVX animals also displayed an increased presence of endomysial distension

when compared CL TA muscles from OVI animals. Lastly, ovariectomy and LCs,

resulted in TA muscles from OVX animals showing an increased endomysial

infiltration after both 20 and 40 LCs. These findings suggest estrogen deprivation

may facilitate an increased level or extent of muscle damage.

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4.5.3 The Muscle Cellular Stress Response Following Ovariectomy and/or Lengthening Contractions When compared to TA muscle from OVI animals, the constitutive expression

of TA muscle Hsp72 but not Hsp25 was decreased in OVX animals. In addition, TA

muscle Hsp72 content in the lengthening contracted TA muscles from OVX animals

was blunted in comparison to TA muscles from OVI animals. However, both TA

muscles from OVI (20 LCs) and OVX (20 and 40 LCs) animals were capable of

increasing Hsp25 content following LCs. These data suggest estrogen loss or

deprivation may selectively down regulate the expression of specific Hsps, thereby

decreasing the ability of muscles from OVX animals to mount a cellular stress

response following a single bout of LCs.

83

Chapter V: Discussion 5.1 Overview

The connection between menopause (estrogen health) and decreased energy

expenditure (Poehlman et al., 1995) may be related to a loss of metabolically active

muscle tissue. Menopause combined with decreased physical activity acts to elevate

central adiposity, oxidative stress and inflammatory markers associated with

sarcopenia (Sites et al., 2002; Bauer and Sieber, 2008; Maltias et al., 2009). Exercise

may help to attenuate many of the negative consequences associated with the

transition of menopause on body composition, physical fitness and overall health

(Roussel et al., 2009).

This study sought to determine the role of estrogen deprivation

(ovariectomy) on the CSR and muscle damage following maximally controlled

stimulated lengthening contractions of rodent TA muscles. It was hypothesized that

TA muscles from OVX animals would show a greater loss of muscle torque following

LCs relative to OVI animals. In addition, morphological markers of muscle damage

were expected to increase in TA muscles from OVX animals when compared to OVI

animals. Lastly, Hsp content (Hsp25 and 72) was expected to be elevated in TA

muscles from OVX animals following LCs.

When physiological measures of muscle function were compared this study

showed no contractile differences in skeletal muscle were observed between TA

muscles from OVI and OVX animals. Thus, estrogen deprivation, at least in the short-

term does not appear to compromise muscle function. However, TA muscles from

OVX had increased immune cell infiltration following muscle damage, suggesting the

84

muscles from OVX animals may be more susceptible to damage. TA muscle Hsp25

content was increased in OVI (20 LCs) and OVX (20 and 40 LCs) animals; however,

TA muscle Hsp72 content was only increased following 40 LCs in OVI animals. This

suggests estrogen deprivation (ovariectomy) may alter the muscle CSR. A decreased

Hsp72 expression was found in the constitutive TA muscles from OVX animals.

Taken together, these findings suggest certain changes in skeletal muscle may

commence shortly after estrogen deprivation occurs. Although, short-term estrogen

deprivation does not appear to effect skeletal muscle torque, the histological and

biochemical responses to LCs appear to be altered following ovariectomy. In the TA

muscles of ovariecotmized animals, an increased immune cell infiltration and an

inability to increase Hsp72 content following 20 and 40 LCs was observed. This

suggests as few as 20 LCs may be a sufficient stress to invoke muscle damage. In

OVX animals the CSR is altered such that skeletal muscle is unable to cope with the

demands of exercise, possibly resulting in additional muscle damage.

5.2 The Anatomical and Physiological Changes Following Ovariectomy

The TA muscles from ovary-intact and ovariectomized animals were

examined three weeks after surgical removal of the ovaries to ensure early changes

in anatomical characteristics had stabilized prior to being subjected to LCs. At the

time, ovariectomy resulted in an 18% increase in body mass and a 20% increase in

TA muscle mass. In addition, uteri mass was decreased by 70% and serum 17-β

estradiol concentration was also significantly decreased in OVX animals. These

85

changes suggest removal of ovaries substantially decreased estrogen and likely

resulted in the alterations to these anatomical parameters.

In agreement with this, Fisher et al. (1998), Christgau et al., (2004) and

Fontenelle et al. (2013), showed increases of 22%, 17.6% and 13.4% in body mass

of OVX animals, respectively. Ovariectomized animals have shown decreased cage

activity (Fisher et al., 1998) and increased food intake (Wade, 1972), resulting in a

positive energy balance (Fisher et al., 1998), thereby becoming more sedentary in

nature. This may also contribute to the increased body mass. In addition, Fisher et

al. (1998) also observed an increase soleus masses by 18% in OVX rats. These

studies support the observation of an increased TA mass in OVX animals observed

herein. Thus the current findings are in agreement with previous studies suggesting

estrogen deprivation results in an increased body mass.

With regards to the uterus, Christgau et al. (2004) and McCormick et al.

(2004) found uteri mass was significantly decreased by 69.2% and 65% in OVX

animals. These values are similar to decreases observed in the present study. These

data support the current findings of increased body and muscle mass and decreased

uteri mass following ovariectomy. Thus, the present study is consistent with other

studies showing estrogen deprivation via ovariectomy results in specific anatomical

changes.

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5.3 Skeletal Muscle Contractility

5.3.1 Tibialis Anterior Muscle Torque

To determine if ovariectomy altered skeletal muscle torque, TA muscles from

both OVI and OVX animals were subjected to 20 and 40 LCs. In general, no

differences in contractile measures were observed in TA muscle torque between

OVI and OVX animals during or following either 20 or 40 LCs. Following 20 LCs, TA

muscle MTT from OVI animals recovered a greater percentage of pre-MTT

compared to OVX animals, suggesting estrogen deprivation may impair skeletal

muscle recovery following LCs.

In humans, studies have observed an estrogenic affect in the maintenance of

skeletal muscle mass and torque (Greeves et al., 1997; 1999; Dionne et al., 2000;

Meeuwsen et al., 2000). In contrast, 60 days after ovariectomy OVX mice generated

less contractile tension, possibly due to an altered interaction between actin and

myosin (Moran et al., 2006). Moran et al. (2007) observed up to a 20% loss in

muscle torque in OVX mice following 30 days of estrogen deprivation. The lack of

any difference between OVI and OVX animals in the current study supports Wohlers

et al., (2009) where no difference in MTT was observed. The lack of observable

differences in TA muscle torque between OVI and OVX animals, in our hands,

suggests the role of estrogen on skeletal muscle torque is minimal.

The results of estrogen on skeletal muscle loss are mixed. In humans, studies

have observed an estrogenic effect on the maintenance of skeletal muscle mass and

torque (Greeves et al., 1999; Dionne et al., 2000; Meeuwsen et al., 2000). However,

animal models tend to shown no differences in skeletal muscle mass between OVI

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and OVX (Moran et al., 2006). The exact reasons for these differences remain

unclear but may be related, in part, to a variety of factors, such as the length of

estrogen deprivation, and fibre type. In the current study, the lack of differences

may be due to the short period of estrogen deprivation (three weeks), the

exercise/stimulation protocol used, the fibre type of the muscle examined (slow vs.

fast twitch). Fridén et al. (1983) observed type II fibres have narrower z-lines,

reflecting fewer attachments for thick and thin filaments (Yamaguchi et al., 1985).

Type II fibres contain fewer sarcoplasmic proteins, myomension and nebulin, which

play roles in sarcomere assembly (Agarkova et al., 2004; Pardo et al., 2005). The TA

muscle is a predominately type II or fast twitch muscle containing very few (<5%)

type I fibres (Locke et al., 1991). Thus, a relatively greater stress is applied to the

cytoskeleton of type II fibres, which may contribute to their greater susceptibility in

fibre damage (Choi, 2014). Estrogen deprivation has been shown to reduce the

relative amount of type IIX muscle fibres (Piccone et al., 2004). The age of animals at

the time of ovariectomy, or species differences may also explain the differences in

skeletal muscle torque.

5.3.2 Maximal Tetanic Tension and Twitch Tension Recovery Following Lengthening Contractions To gain an understanding of potential differences in skeletal muscle recovery

in an estrogen deprived state, TA muscle MTT and MTw were measured prior to,

directly after (t=0) and 10-minutes following LCs in OVI and OVX animals. Ten

minutes following the last (20th) LC, MTT tetanic tension of the TA muscles showed

a significant decrease in OVX animals when compared to pre-MTT. However,

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muscles from OVI animals at ten minutes after the 20th LC had significantly

recovered from pre-tetanic tension. This suggests estrogen may either minimize the

loss of MTT following LCs and/or facilitate recovery following LCs. A similar

response has been observed in aged rat muscle, where maximal tetanic force was

decreased and a ~60% decrease in recovery from intermittent isometric

contractions (Degens and Always, 2003). Thus, OVX animals may be similar to an

“aged” condition, in this respect.

Estrogen deprivation may alter MTT and MTw through multiple mechanisms

including due to interference with Ca2+ binding sites (Martinez-Azorin et al., 1992).

Diethylstilbestrol, an endocrine disruptor, directly inhibited rabbit muscle

sarcoplasmic reticulum Ca2+ ATPase by interfering with Ca2+ binding sites

(Martinez-Azorin et al., 1992). Estrogen deprivation may affect Ca2+ reuptake into

skeletal muscle, indicated by an increase in soleus half-relaxation time (McCormick

et al., 2004). Ovariectomy may also result in decreased fast myosin heavy chain

expression observed in the soleus muscle (Kadi et al., 2002), indicating the estrogen

deprivation may reduce contraction speed (McCormick et al., 2004).

Using an in vitro model, McCormick et al. (2004) showed that time to peak

tension was decreased in OVX animals, although maximal isometric torque

remained unchanged in the soleus from OVX animals. In addition, using a similar

model, after three weeks of ovariectomy, Hubal et al. (2005) observed no significant

difference in peak isometric torque in TA and EDL muscles from OVX mice and sham

mice; however, peak isometric torque tended to be ~5-10% lower in OVX mice.

Furthermore, using an in situ model Fisher et al., (1998) found no significant

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differences in peak tetanic tension in the EDL and soleus of OVX rats with a two-

week period of ovariectomy. Similar to the current study, in vivo or in situ models of

exercise do not appear to alter MTT following ovariectomy. However, in vitro

measures of contractility resulted in decreased skeletal muscle MTT. Using a three-

week period of estrogen deprivation, Warren et al. (1996) showed an 18% decrease

in in vitro pre-peak isometric torque in EDL muscles from OVX mice, although no

significant difference was observed in post-peak isometric tension in EDL muscles

from OVI and OVX mice. Taken together, these differences in MTT observed may be

the result of different models of muscle damage used (in vivo, in vitro and in situ), as

well as the length of estrogen deprivation prior to contractile measures.

In the present study, TA muscle MTw was examined with no differences

observed between OVI and OVX animals. Bunratsami et al. (2015) showed twitch

tension was significantly decreased in EDL muscles from OVX rats. Using an in vitro

model, isometric MTw in the EDL was significantly decreased in OVX mice compared

to sham mice (Lai et al., 2016). Similar to MTT, mixed results were observed in MTw

between OVX and sham animals from the current study when compared to past

results. The differences in the present study to others may again be the model (in

vivo versus in vitro) to determine MTw. Furthermore, neither Bunratsami et al.

(2015) or Lai et al. (2016) employed an exercise protocol to induce muscle fatigue

or damage. In contrast, the present study examined the role of ovariectomy on

skeletal muscle torque using both 20 and 40 LCs.

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5.3.3 The Active Component of Tibilais Anterior Muscle Torque

The active component of muscle torque is a measure of tetanic tension prior

to the foot pedal actively lengthening the TA muscle and peaks between 0-0.2ms of a

LC. No differences were observed in the active components of TA muscle torque

between OVI and OVX animals. To date, no literature is available on the active

component of skeletal muscle torque measured during in vivo contraction. In the

closest study available, Moran et al. (2007) used 4 weeks of estrogen deprivation

and showed a 10% decrease (in vitro) in the active stiffness in the soleus, which is

predominantly a slow oxidative muscle. Whether differences between this finding

and the present study are due to in vivo versus in vitro muscle contraction, or some

other factor(s) remains to be determined.

5.3.4 The Passive Component of Tibialis Anterior Muscle Torque

The passive component of torque refers to the torque generated after MTT

has plateaued. This reflects the stretch of the muscle generated by the tension (pull)

from the foot pedal moving and occurs between 0.3ms to 1.0 seconds. The passive

component of torque may be considered as an indirect measure of muscle and

connective tissue resistance to stretch. The passive component of TA muscle torque

decreased in OVI animals prior to TA muscles from OVX animals throughout both 20

and 40 LCs, suggesting tissues from OVX animals may be stiffer or be beginning to

show an increased stiffness compared to connective tissues from OVI animals.

Moran et al. (2007) examined the (in vitro) soleus (passive) stiffness in OVX mice

and showed it increased by 7.7% after four weeks of ovariectomy. Skeletal muscle

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connective tissue is essential for the transmission of force and stability throughout

skeletal muscle structures (Kjaer, 2004). An increased collagen formation has been

reported in articular cartilage from ovariectomized rats (Fontenelle et al., 2013).

Estrogen deprivation has been shown to increase collagen production, thereby

increasing ossification and decreasing mechanical resistance (Fontenelle et al.,

2013). Taken together, the observations and the present work suggest estrogen

deprivation may result in an increased connective tissue stiffness and a slow but

progressive loss in the passive component of muscle torque.

In the present study, no differences in the passive component of TA muscle

torque were observed between OVI and OVX animals. However, a trend towards an

elevated passive component of TA muscle torque was observed in OVI animals

(compared to OVX animals) suggesting a decreased elasticity in connective tissues

may have commenced. The passive component of TA muscle torque from OVI

animals decreased before TA muscles from OVX animals. The production of passive

tension and storage and release of elastic recoil energy in skeletal muscle may be

necessary for energy transmission in LCs (Lindstedt et al., 2001). Tendons are

capable of returning 85-95% of the stored energy (Shadwick, 1990; Matson et al.,

2012). A stiffer spring may enhance the amount of elastic recoil energy available in

the stretch shortening cycle (Komi, 2000). Thus the increase in articular cartilage in

OVX animals may allow for greater energy transmission allowing for prolonged

torque production and may enhance the “spring like” (Lindstedt et al., 2001)

property of the muscle. Seyfarth et al., (2000) showed an increased muscle stiffness

(tighter muscle spring) allowed for improvements in lengthening exercises, such as

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jumping. Taken together, estrogen deprivation may result in increased tissue

stiffness allowing for greater transmission of energy.

5.4 Histological Characteristics

5.4.1 Muscle Fibre Area and Circularity in Ovary-Intact and Ovariectomized Animals

Estrogen deprivation increases body mass, which would account for the

increased fibre area observed since it would require a greater muscle mass to cope

with the increased body mass. Following ovariectomy, TA muscle fibre area of the

CL limb was increased in OVX animals when compared to the CL limb of OVI

animals. Given that both body mass and skeletal muscle mass were increased

following ovariectomy, the increased body mass observed herein may account for

the observed increase in CL TA muscles fibre area from OVX animals. Fibre

circularity (a measure of a perfect circle) was also increased in the TA muscles from

OVX animals following both 20 and 40 LCs. In contrast, TA muscles from OVI

animals only showed an increase in fibre circularity after 40 LCs. Although the

absolute stress of LCs was the same for both OVI and OVX animals, the different

response observed between the two suggests 20 LCs was an insufficient stress to

invoke fibre area and circularity increases in OVI animals.

5.4.2 Endomysial Infiltration

Using the visual scale developed by Rizo-Roca et al. (2015) H&E stained

muscle fibres were examined for damage by light microscopy. An increase in the

infiltration of immune cells was observed in the endomysium of TA muscle fibres

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from OVX animals following both 20 and 40 LCs. In contrast, endomysial infiltration

was only increased following 40 LCs in TA muscles from OVI animals. Similar to

fibre circularity, this suggests muscles from OVX animals may be more susceptible

to the stress imposed by LCs. Twenty LCs may be experienced as a relatively greater

stress in OVX animals. It appears that the changes due to estrogen deprivation may

alter either the permeability of connective tissue or the activity of immune cells.

Given that LCs require the absorption of energy, a muscle incapable of this action

may be more easily damaged (Roberts, 2016).

Neutrophil and macrophage infiltration are known to be sensitive to ovarian

hormone (progesterone and estrogen) concentrations (Zuckerman et al., 1996;

Angstwrum et al., 1997; Komulainen et al., 1999; Tiidus et al., 2001, 2005). McClung

et al. (2007) observed increased neutrophil and macrophage invasion in the soleus

muscle from OVX animals during the initial week of recovery from disuse injury.

Multiple studies using downhill treadmill running have shown estrogen attenuates

neutrophil infiltration (St. Pierre-Schneider et al., 1999; Enns and Tiidus, 2010;

Nedergaard et al., 2013). The present work suggests estrogen deprived skeletal

muscle may not recover from the injury induced by LC-biased exercise. Similarly,

Tiidus et al. (2001) showed an estrogenic effect on post-exercise neutrophil

invasion, suggesting estrogen mediated leukocyte infiltration following exercise and

may result in a reduction of intramuscular leukocyte infiltration (Iqbal et al., 2008).

Thus, following LCs, TA muscles from OVX animals are likely to experience greater

muscle damage when compared to TA muscles from OVI animals.

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Whether estrogen attenuates the primary or secondary phase of muscle

damage through the localized inflammatory response remains to be determined

(Savage and Clarkson, 2002). Stupka and Tiidus (2001) examined neutrophil

infiltration following ischemia-reperfusion injury in the hind limb of female rats and

showed OVX rats exhibited an increased myeloperoxidase activity when compared

to OVI animals. In addition, Iqbal et al. (2008) observed an increased neutrophil and

macrophage invasion in the soleus and vastus lateralis muscles from OVX rats

following downhill treadmill running. These data suggest estrogen attenuates

leukocyte infiltration following injury or exercise. Whether estrogen acts in

conjunction with Hsps or other cellular mechanisms, thereby providing resistance

against muscular damage, to limit leukocyte infiltration remains to be determined.

5.4.3 Endomysial Distension (Between Ovary-Intact and Ovariectomized Animals)

Endomysial distension refers to a distended space in the connective tissue

between the individual muscle fibres. Not surprisingly, TA muscles from OVI

animals showed significant increases in endomysial distension following both 20

and 40 LCs. This finding may be due to an increase in connective tissues in TA

muscles from OVX animals. In the aged animal, Nielsen et al. (1998) has shown

increased stiffness in the TA tendons of aged rats and better energy-absorption

(Nielsen et al., 1998). Similar to aged animals, OVX animals show increased articular

cartilage (Fontenelle et al., 2013). Thus, the increased connective tissue may allow

for greater energy absorption and possible torque transmission. The increased

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tensile stiffness in OVX animals may explain the decreased distension or stretch in

connective tissue from OVX animals.

5.5 The Cellular Stress Response Following Ovariectomy and/or Lengthening Contractions Increased levels of Hsp27 and 70 have been reported in human skeletal

muscle following high-force ‘eccentric’ exercise (Reichsman et al., 1991; Thompson

and Scordilis, 1994; Febbraio and Kokoulas, 2000; Feasson et al., 2002; Thompson

et al., 2002; Thompson et al., 2003; Willoughby et al., 2003). Whether increased Hsp

levels are indicative of the response to skeletal muscle damage or an attempt to

prepare against (protect) future stressful events is unclear. However, estrogen may

be involved in the regulation of Hsp expression following damaging exercise.

The significant decrease (57%) of constitutive expression of Hsp72 in TA

muscles from OVX animals observed in the present study is supported by other

findings. Bombardier et al. (2013) observed similar decreases in the constitutive

expression of Hsp70 in the soleus muscles from OVX animals. Sexual dimorphism of

the Hsp72 response to a variety of stressors is well-documented and following

stress the up-regulation of Hsp72 appears to be partially mediated by estrogen

interacting with the sympathetic nervous system (Nickerson, 2006). Since the

constitutive Hsp72 expression level may be necessary for skeletal muscle to mount

an immediate cellular stress response, any observed decrease in constitutive

expression of Hsp72 in OVX animals may result in a reduced ability to protect

against skeletal muscle damage. In support of this, McArdle et al. (2004) observed

mice expressing Hsp70 showed increased protection from secondary damage three

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days following LCs. McArdle et al. (2004) suggest Hsp70 might be a necessary

component of cellular repair machinery, indicating that the presence of Hsp70 may

help repair skeletal muscle more readily following LCs. This may also augment an

increased immune cell or inflammatory response following LCs. The observed

presence of an increased endomysial infiltration of immune cells in TA muscles from

OVX animals following 20 and 40 LCs supports this idea. Hsps may also function as

damage associated molecular patterns (DAMPs) that may be released from damaged

cells under necrotic conditions (Broere et al., 2011). If the constitutive expression of

Hsp72 is decreased it may result in a decreased DAMPs signal when a fibre is

damaged, thus decreasing any subsequent repair responses. Taken together, the

decrease in Hsp72 may result in a decreased capacity of OVX animals to protect

skeletal muscles against damage.

This study also observed an increased TA Hsp25 content for both OVI and

OVX animals following 40 LCs whereas only Hsp25 increased in the TA muscle from

OVX rats following 20 LCs. The lack of increase in TA muscle Hsp25 content in OVI

animals following 20 LCs suggests the stress was insufficient to warrant an Hsp

response. Conversely, the increased Hsp25 expression in OVX animals suggests

estrogen deprivation does not affect Hsp25 expression.

A ‘critical amount’ or threshold of Hsp72 has been suggested to be necessary

to provide protection (Locke and Tanguay, 1996). Thus, the decreased constitutive

expression of Hsp72 TA muscle may thereby prevent or not allow an adequate CSR

response. Thus, the decreased constitutive Hsp72 expression may lead to the loss in

the capacity to cope with the stressors of daily life. It should be noted that an

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increased number of necrotic fibres was observed in CL TA muscles from OVX

animals. Therefore, the decreased constitutive expression of TA muscle Hsp72

content in OVX animals may result in the inability to handle the stressors of

everyday life, resulting in an increased presence of necrotic fibers observed in the

CL TA muscles from OVX animals.

5.5.1 Differences in Heat Shock Protein 25 and 72 Content Following Lengthening Contractions Heat shock proteins act as protein chaperones ensuring the correct folding

refolding of damaged proteins from stressful events. Hsp72 content is known to

stabilize both the structure and function of the sarcoplasmic reticulum Ca2+ pump in

muscle subjected to heat stress (Tupling et al., 2004; Fu and Tupling, 2009). Hsp25

predominately binds to cytoskeletal/myofibrillar proteins, such as desmin, α-

actinin, actin and myosin, possibly protecting them from the stress of converting

energy through a structure (Hernado and Manso, 1997; Atomi et al., 2000; Koh and

Escobedo, 2004; Paulsen et al., 2009). In mouse models, Hsp25 has been found to

accumulate at the z-disk and within intermediate structures, (possibly desmin [Koh

and Escobedo, 2004]) following damaging eccentric exercise (Larkins et al., 2012).

In addition, some of the Hsp25 present in the ‘unstressed’ muscle fibres is localized

within the sarcolemma (Larkins et al., 2012), suggesting different functions for

Hsp25 and Hsp72. Given that Hsp25 content was over expressed in OVX animals

following 20 LCs, it underlines its potential importance in protecting skeletal muscle

from mechanical insult. These data suggest Hsp25 and Hsp72 may have different

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protective mechanisms, possibly resulting in the differences observed in expression

between Hsp25 and 72.

5.5.2 The Constitutive Heat Shock Protein 72 Expression in the Tibialis Anterior

Skeletal muscle fibre types may play a role in the observed differences for

Hsp72 content. The TA muscle is comprised primarily of type II fibres. Locke et al.

(1991) observed decreased constitutive expression of Hsp72 in the TA muscle in the

regions expressing primarily type IIB fibres. In support of this, Bombardier et al.

(2009) observed that independently of hormone treatment type II fibres showed

decreased Hsp70 expression compared to type I skeletal muscle fibres. This could

suggest that type II fibres may be subject to increased damage as any further loss of

Hsp72 content may result a decreased capacity to protect skeletal muscle following

exercise.

In aged cells and tissues a decreased Hsp content results in a decreased

protection. Vasilaki et al. (2002) observed a diminished Hsp response in the muscles

of elderly rodents. Using a transgenic Hsp70 overexpression mouse model and LCs

to induce muscle damage, McArdle et al. (2004) showed muscles from Hsp70

transgenic mice had less fibre damage when compared to wild type mice. The

decreased constitutive TA muscle Hsp72 expression observed following

ovariectomy may render the muscles from OVX animals more susceptible to usage

or stress.

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5.5.3 Heat Shock Factor 1 Content in Ovariectomized Animals

HSF1 content mediates the stress induced up-regulation of Hsps (Yasuhara et

al., 2011). Yasuhara et al. (2011) showed soleus regrowth following a four-week

recovery was partially inhibited in HSF1-null mice HSF1. Interestingly, this was not

the case for Hsp25. The presence of HSF1 content may be necessary for the up-

regulation of Hsp72 content. There may be a ‘critical level’ in the ‘baseline

expression’ of Hsps, thereby influencing the regulation of skeletal muscle mass

(Yasuhara et al., 2011). The observed difference in Hsp25 and 72 content following

20 and 40 LCs in OVI and OVX animals may be an indication of their different

regulatory mechanisms (Yasuhara et al., 2011). This suggests Hsp25 expression may

be increased by factors other than HSF1, given the lack of Hsp25 increase observed

following 20 or 40 LCs in OVI animals. The p38 mitogen-activated protein kinase

pathway is activated by stress and also plays an important role in the immune

response, as well as in the regulation of cell survival in differentiation (Cuadrado

and Nebreda, 2010). p38 is involved in the balance of protein synthesis and

degradation (Evertsson et al., 2014). p38 has been identified as a likely mediator of

catabolic signaling in skeletal muscle (Tracey, 2002) and Hsp25 is phosphorylated

through the p38 pathway (Stokoe et al., 1992). Thus, Hsp25 may function through

the p38 pathway to maintain protein integrity following muscle damage.

5.5.4 Heat Shock Protein 72 Content in Ovariectomized Animals

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In the current study, TA muscle Hsp72 content in the lengthening contracted

limb was not increased following both 20 and 40 LCs and thus OVX animals

appeared to similar to an “aged” like condition. Hsps have been shown to protect

cells and tissues, including skeletal muscle; however, tissues from aged animals

show a decreased Hsp content and therefore a diminished protection (Blake et al.,

1991; Heydari et al., 1993; Nitta et al., 1994; Locke and Tanguay, 1996). For

example, following exercise, when hearts from adult and aged animals were

examined, the hearts from aged animals showed a decreased HSF1 activation, Hsp72

content and myocardial protection (Locke and Tanguay, 1996). Whether the same

lack of protection occurs in OVX animals remains to be determined.

The stress-inducible transcription of Hsp72 and other Hsps are mediated by

HSF1 (Amin et al., 1988). In the current study, although HSF1 expression was not

significantly reduced in TA muscles from OVX animals, a decreased trend was

observed. The finding of non-significance in HSF1 content may be due to the lack of

power (n=4). Thus, it follows that if HSF1 is decreased then the ability to increase

Hsp72 content may also be attenuated. If ovariectomy (estrogen deprivation) alters

HSF1 content similar to aged cells and tissues, then ovariectomy may render the

cells and tissues more susceptible to stressors due to their decreased ability to

protect cells and tissue from damage.

Yasuhara et al. (2011) showed that in HSF-null mice, Hsp72 content was

significantly decreased when compared to wild-type mice. A similar trend of

decreased HSF1 was observed in the present study in TA muscles from OVX animals.

In view of this, skeletal muscle from OVX animals may be unable to translate the

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stress signals into the biochemical responses necessary to invoke the protective

stress response. Thus, it may be the case that a decreased CSR observed with OVX

animals may result in a decreased ability to protect skeletal muscle from LCs,

thereby increase muscle damage.

5.6 Connections to Post-Menopausal Females

These findings support the well-established fact that estrogen deprivation

results in increased body mass. In addition, females in the early phase of menopause

may not show changes in skeletal muscle performance (Sewright et al., 2008). Post-

menopausal females will experience the loss of elastic properties resulting in an

increased connective tissue stiffness. Furthermore, following a single bout of

moderate LCs (20 LCs) menopausal females may be more likely to exhibit a localized

inflammatory response, as observed by increased endomysial infiltration of immune

cells in TA muscle following both 20 and 40 LCs in OVX animals. This suggests, that

even moderate forms of exercise may result in an increased inflammatory response.

Lastly, the decreased Hsp72 content following LCs suggests that similar to aging,

menopausal females may have a decreased stress response following a single bout

of LCs. Thus, since menopausal females may have decreased cellular and tissue

protection following a single bout of mild or moderate LCs, females initially

beginning exercise should avoid excessive exercise and begin gradually until the

muscles have adapted. Given the daily stressors of life, such as stair descending

involve LCs, skeletal muscle of menopausal females may have a decreased capacity

to mount a CSR, thus decreasing muscles protection following daily activities or

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exercise. Whether this contributes to the onset, or an earlier onset of sarcopenia

remains to be determined.

5.7 Conclusions

This study observed no difference in TA muscles contractile properties

between OVI and OVX animals following both 20 and 40 LCs. An increased immune

cells presence was detected in TA muscles from OVX animals. A decreased

constitutive TA muscle Hsp72 expression in OVX animals, as well as, no elevation in

Hsp72 content in TA muscle from OVX animals following LCs was observed.

However, Hsp25 content was increased in OVX animals following 20 and 40 LCs.

This work suggests during the early phases of menopause changes to the CSR may

occur prior to contractile changes. Post-menopausal females may not be able to

mount an adequate cellular defense to a low number of LCs.

5.8 Limitations

The effects of estrogen deprivation on skeletal muscle after LCs were

examined. In the current study the period of estrogen deprivation lasted three

weeks prior to the LC procedure. Three weeks of estrogen deprivation may not have

been sufficient time to observe long-term alterations in TA muscle torque between

OVI and OVX animals. Thus, future studies should examine the effects on a longer

term of estrogen deprivation on skeletal muscle. On average, the lifespan of a rat is

approximately two years; in the current study OVX animals were estrogen-deprived

following surgical removal of the ovaries and prior to LCs for only 3 weeks. In the

life of a rodent, this time is approximately equivalent to 2.4 years of a human

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lifespan. Thus, it is possible that short-term estrogen deprivation may not

immediately cause the differences in muscle contractility, as some alterations may

take longer to develop. In view of this, long-term estrogen deprivation may be

necessary to observe significant estrogen related changed to take place/occur in

skeletal muscle torque and markers of muscle damage.

An enzyme linked immunosorbent assay was used to measure 17-β estradiol

serum concentrations. Serum 17-β estradiol concentration from OVX animals was

only reduced by ~48%. While it remains possible that estrogen was decreased. A

likely explanation for the less than half decrease in serum 17-β estradiol is the

antibodies used bound to other molecules, including the two other forms of

estrogen (i.e. estrone and estriol) or other mineralcorticoids such as testosterone

may have also bound to the epitope resulting in non-specific binding. Also,

menstruating rodents go through four phases of the estrous cycle (proestrus, estrus,

metestru and diestrus). These phases are associated with varying levels of 17-β

estradiol, with highest levels during proetsrus. Thus, non-specific binding or varying

stages of estrous may account for the levels of serum 17-β estradiol concentrations

measured in OVX animals when compared to the blood serum of OVI animals.

Although the effects of estrogen were examined in skeletal muscle

contractility and muscle damage, this study did not account for the effect of

progesterone on skeletal muscle. Progesterone concentrations were not measured

and thus, the effect of circulating progesterone on skeletal muscle cannot be

accounted for in the current study. Whether the removal of estrogen was

compensated by an increase in progesterone remains unlikely.

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Since morphology was only examined 24 hours after LCs it is not possible to

determine the role of estrogen plays in endomysial infiltration in the days following

LCs and thus the role of estrogen in skeletal muscle recovery cannot be examined. In

addition, when skeletal muscles were examined the investigator was not blinded,

potentially resulting in some bias in measurements. Lastly, this study examined

medial sections of skeletal muscle by Western blotting. Thus, it is not possible to

determine if the Hsp response observed was localized to that area of the skeletal

muscle or throughout the entire muscle.

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Chapter VII: Appendix 7.1 Tissue Preparation and Sectioning Skeletal Muscle Materials:

Tempered glass plate Single edged blade (Personna American Safety razor Co., Cat #: 94-120-71) 2-18G needles Cut cork sections (~3cm x 3cm) Dewar for liquid nitrogen and Isopentane Suspension apparatus

o Sturdy stand with a vertical bar o Horizontal arm secured by a vertical bar o Thin rope to suspend the metallic bowl

Small metallic bowl (~250mL) Tongs Liquid Nitrogen (~1L) Isopentane (Methylbutane or 3-methylbutane) (~150mL) Optimal Cutting Temperature medium (OCT) (VWR, Cat #: CA95057-838)

Preparation:

1. Label cork pieces on the bottom and side with the subject, condition and muscle.

2. Pour liquid nitrogen into the dewar. 3. Set up the thin rope and metal bowl on the suspension apparatus above the

dewar of liquid nitrogen. 4. Pour isopentate (~150mL) into the metal bowl and lower into the liquid

nitrogen in order for isopentane to cool (the bottom of the metallic bowl will begin to turn white).

Procedure:

1. Raise the metal bowl out of the liquid nitrogen. 2. Section the muscle into 100-150mg pieces by using the both single edged

blades in a scissors manner, cutting perpendicular to the fibres (cross-section).

3. Dab a small dollop of OCT onto the cork and submerse it into the isopentane quickly to solidify the outside layers, while the inside remains gelatinous.

4. Repeat step 3 to build up another layer of OCT. 5. Using the two 18G needles, orient and submerge the muscle section with the

fibres facing directly upwards. 6. Cover the muscle section with OCT and submerge the completed cork and

muscle section into isopentane face down. 7. Remove the muscle section from the isopentane and place in dewar of liquid

nitrogen while preparing other muscle samples.

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8. Remove from liquid nitrogen, place the samples in a plastic container, add some liquid nitrogen to the container and store the samples at -80°C.

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7.2 Tissue and Blood Collection Materials:

1.5mL eppendorf tubes 18G needles Evacuated Collection Tubes (Vacutainer, BD, NJ, USA) heparin and heparin-

free Centrifuge (Beckman Coulter, Model #: Allegra 6R, Serial #: ALR01F81) Surgical scissors Rat toothed tweezers Blunt probe Clamp Plastic cutting board Isoflurane/oxygen gas mixture; 1L/min Oscillator (Lab-Line International Inc., Orbit P4, Model #: 322) Suction pipets

Preparation:

1. Label eppendorf tubes on the top and side with the subject, condition and muscle.

2. Label evacuated collection tubes with the subject and blood sample. 3. Turn on the centrifuge and set to 4°C.

Procedure:

1. Under anesthesia pick up the rodent’s abdomen with the rat toothed tweezers and cut through the abdomen and diaphragm using the scissors.

2. Extract the blood from the heart using an 18G needle and place half the blood sample in the evacuated collection tubes containing heparin and heparin-free tubes.

3. Place the blood samples on the oscillator for 15 minutes at room temperature and than store in the fridge at 4°C on an oscillator until ready for centrifuge.

4. Pull away fur and skin on the rodent’s treatment limb and use fine scissors to cut away at the epimysium.

5. Use a blunt probe to tear away the epimysium at the top near the patella in order to free the tibilais anterior.

6. Clamp the tendon at the bottom neat the foot and cut the tendon with fine scissors, using the clamp to gently pull the muscle away.

7. Use the fine scissors to cut the tibilais anterior at the top of the knee.

8. Place in a small weight boat the weight the muscle before sectioning for analysis.

9. Repeat steps 4-8 for the contra-lateral control limb. 10. Following collection of all rodent tissue, place the evacuated collection tubes

in the centrifuge, making sure they are balanced.

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11. Centrifuge the blood samples at 4°C for 15 minutes, at 3,000rpm. 12. Gently use the suction tubes to extract the plasma and serum and place them

into pre-labeled 1.5mL eppendorf tubes and store at -80°C for later analysis.

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7.3 Muscle Homogenization Materials:

Bead Bug Microtube Homogenizer (Model D1030, Product Code: 1143051039)

Acid Washed, Stainless Steel Prefilled Tubes (2.8mm diameter) (Benchmark, Item #: D1033-28)

RIPA buffer (Sigma Aldrich, Cat #: R0278) Protease inhibitor (Sigma Aldrich, Cat #: P8340) Ice bucket for samples Cut muscle samples (in ice) Centrifuge (Eppendorf, Model #: 5415 C) Eppendorf Pipets Labeled 1.5ml Eppendorf tubes

Preparation:

1. Place frozen muscle samples on ice in order to keep cool. Procedure:

1. Label prefilled tubes to designate appropriate muscle sample. 2. Add ten times the amount of RIAP buffer solution (based on muscle weight)

to the prefilled tube and 0.100μl of protease inhibitor per mg of muscle to prefilled tubes.

3. Place the frozen sample into prefilled tubes with RIPA buffer and protease inhibitor on and then place on ice to keep cool.

4. Repeat step 2 and 3 for all muscle samples. 5. Orient prefilled tubes into the Bead Bug homogenizer the same way, place

the control tube in the uppermost slot and the treatment in the lowest slot, leave the center slot empty.

a. Orientation image 6. Homogenize sample at a set speed of 400, set timer to 45 seconds. 7. After each homogenate, place the prefilled tubes back on ice to cool for

30seconds-1minute. 8. Place the prefilled tubes back into the Bead Bug homogenizer and repeat

steps 6 and 7 two more times. 9. Place prefilled tubes into centrifuge and spin for 30 seconds or longer at

3,000 rpm, to get rid of bubbles and determine if further homogenization is needed.

10. If further homogenization is needed place the prefilled tubes back on ice to allow for cooling for 30 seconds to 1 minute.

11. Repeat step 5-7 two to three more times. 12. Again, place the prefilled tubes into centrifuge and spin for 30 seconds or

longer at 3,000 rpm, to get rid of bubbles and determine if further homogenization is needed.

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13. Once the muscle sample is fully homogenized, extract it with an Eppendorf pipet along the side of the prefilled tubes, in order to avoid the beads and connective tissue at the bottom.

14. Place homogenate into labeled Eppendorf tube and place in -20°C freezer for short-term storage.

15. Once all samples are ground, place all in -80°C freezer for long-term storage.

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7.4 Determination of Protein Concentration—Lowry Assay Lowry-Protein Determination Loaded Sample

ddH2O STD Sample Reagent Phenol

Blank 0.5 ml - - -

20μg 0.48 20μl 5ml 0.5ml

40μg 0.46 40μl 5ml 0.5ml

60μg 0.44 60μl 5ml 0.5ml

80μg 0.42 80μl 5ml 0.5ml

100μg 0.40 100μl 5ml 0.5ml

Samples ddH2O STD Sample 1 0.500ml 5μl 2 0.500ml 5μl 3 0.500ml 5μl 4 0.500ml 5μl 36 0.500ml 5μl Preparation: 1ml (2% weight/volume) CuSO45H20 4ml (3% weight/volume) Na2CO3 in 0.1 N NaOH

In sets of three tubes (16) add 5ml of Lowry reagent = 240ml o Make up 250ml of Lowry reagent

Lowry Reagent

5ml Na-Tartrate 5ml CuSO45H20 240ml of Na2CO3 1. In a beaker, place 240ml of Na2CO3 and add Na-Tartrate first (5ml), than add

CuSO4 (5ml). 2. Add standards and samples together. 3. Add Lowry reagent to tubes than vortex and let rest for 10 minutes. 4. Mix the Phenol reagent with ddH2O, in a 1:2 ratio, i.e., for 45 tubes 70ml is

needed (0.5ml/tube). a. Make up 70ml, i.e., 20ml of phenol in 50ml of ddH2O.

5. Add 0.5ml of phenol reagent to each tube while vortexing tubes. Let each tube sit for 5 minutes before reading.

6. Read the tubes at a wavelength of 660nm (Turner, model # 340).

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7.5 SDS-PAGE, Western Blotting and Development

1. Assemble pouring apparatus for SDS-PAGE, pour gels o Add 1mL of APS to 1mL of ddH2O

2. Load sample with 1:1 ration of sample buffer o Load between 50-100μg per lane o Run the gel between 100-120 volts, until the dye runs off the gel o Ensure bubbles are present o It should take approximately 1.5 hours for the dye to run off the gel

3. While the gel is running gather materials to transfer to Western Blot (large casserole dish) and make transfer buffer.

4. Transfer buffer: 100ml of 10x Running Buffer, 700mL of ddH2O, 200mL of methanol.

o Mix in this order in order to avoid salt loss from the solution. o Equilibrate the gel and nitrocellulose paper for ten minutes and set up

transfer sandwich. o Mark the nitrocellulose: one diagonal for the first well, one square for

the last well. 5. Sandwich: Black part of the apparatusBrillo pad, three filter papers,

Nitrocellulose, white part of the apparatus3 filter papers, Brillo pad, close and lock the apparatus to create the sandwich.

o Repeat for both sandwiches 6. Ensure no bubbles are present, use a test tube to smooth the gel down,

carefully lift the ends of the gel remove bubble. 7. Insert the ice pack and fill with transfer buffer. Run at 60 volts for two hours.

o Ensure bubbles are present before turning on the stir bar o Replace the ice pack every hour

8. Remove blot and block in 5% blotto for one hour or overnight 9. 5% Blotto: 5g of non-fat milk powder, 100mL of TBS 10. Wash blot:

o First wash: 1x TTBS for five minutes o Second wash: 1x TTBS for five minutes

11. Incubate in 2% blotto with primary anti-body oscillating in the fridge for 3 hours or overnight

12. 2% Blotto: 2g of non-fat milk powder and 100mL of TBS 13. Primary anti-body dilution:

o 10mL of 2% blotto o Add 0.010μl of primary anti-body to make a 1:1000 dilution

14. Wash blot in TTBS for two washes at five minutes each and wash once in TBS for five minutes

15. Incubate in 2% blotto with secondary goat anti-rabbit IgG HRP-linked antibody (BioRad Cat# 170-6515) anti-body for 1 hour at room temperature on tectator (Lab-Line Instruments Inc., model #: 3520)

16. Secondary anti-body dilution: o 20mL of 2% blotto o Add 0.020μl of secondary anti-body (goat anti-rabbit HRP)

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17. Place in solution developer o Add 8ml of Luminata Forte Western HRP Substrate (Millipore

Corporation, Billerica, MA, Cat #: WBLUF0100) o Incubate each blot separately for 5 minutes.

18. Place blot face down on Li-Cor C-digit IS version 3.1 (Mandel Scientific, model #: CDG-001073 3600).

19. Scan image at “standard” sensitivity for six minutes, using Image Studio Digits (version 5.2.5)

20. Select best image and quantify lanes and safe quantified values in an Excel file.

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7.6 Enzyme-Linked Immunosorbent Assay (ELISA) Materials:

17 beta Estradiol ELISA Kit (Abcam, ab108667) Orbit Environ Shaker (Lab-Line Instruments, Inc., Model #: 3527) Deionised distilled water Serum samples Kimtech wipes Aluminum foil

Preparation:

Bring ELISA kit to room temperature. Place serum samples on ice to thaw. Turn on incubator and set to 37°C. Make wash solution by adding 50ml of Wash Solution to 450ml of ddH20.

Procedure:

1. Add 25μl of standards, control and sample to appropriate wells. 2. Add 200μl of 17β-Estradiol HRP Conjugate to each well. 3. Cover 96 well plate with aluminum foil and incubate at 37°C for two hours. 4. Remove the well contents and wash the each well three times with 300μl of

Wash Solution each time. a. Blot excess solution from wells following last wash.

5. Add 100μl of TBM Substrate to each well and incubate for exactly 30 minutes at room temperature.

6. Add 100μl of Stop Solution to each well and read the 96 well plate at a wavelength of 450nm within 30 minutes.

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7.7 Muscle Cross-Sectioning Materials:

Croystat, Myotome (Cryo-Cut, American Optimal Company, model #: 830) Honeycomb block/chuck Single edged blade (Personna American Safety razor Co., Cat #: 94-120-71) Microscope slides (stored at room temperature) (VWR, Cat#: CA48323-190) Paint brush (Chinese boar bristles, 1/4 inch, flat or bright, cut at a 45 degree

angle) OCT compound (VWR, Cat #: CA95057-838) 70% Ethanol 95% Ethanol Slide rack Kimtech wipes

Preparation: 1. Turn the cryostat and, place a single edged blade in the cryostat and the

apparatus to reach -20°C. 2. Take the cryostat blade out of the -20°C freezer and position the blade inside the

cryostat. Tighten the furthest knob to the left. 3. Remove the sample being sectioned from the cork using the cooled single edged

blade and bond the section to the chuck with a small amount of OCT compound. To fully bond leave the chuck inside the cryostat for 10-15 minutes.

4. Trim and remove excess OCT compound from the periphery of the sample into a triangle shape, with the most arrow part being the first cut (pointed up).

5. Insert the chuck into the holder and tighten. Procedure: 1. Adjust the angle of the blade to 4° with the black knob closets to you (may be

done). 2. Adjusting the knob behind the curved ruler at the back of the cryostat in order

to just the section thickness (10-12μm). 3. Adjust the blade cart height by locking and unlocking the lever closest to you. 4. Cut off a few sections in order to get nice consistent slices and ensure the blade

is cutting properly. 5. Crank the black hand wheel on the right of the machine to cut sections. Ensure

that the cutting is even and not curling, folding or tearing (caused by crevasses in the blade).

A. Ensure that the axel does not become unhinged after numerous sections. B. Reset the axel by using the knob at the front of the machine and readjusting

the blade. 6. Hold the slide on the frosted end and place the frosted end down towards the

blade. 7. Adhere the slide by using a room temperature slide and angling it at a 45° angle

above the blade. Slowly move the slide towards the section and allow it to

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adhere. A. Minimize drying time, as the tissue will undergo alterations and it warms

and dries. 8. Once the section has adhered to the slide, submerse in fixation solution (95%

ethanol) and than add it to the slide rack while cutting the rest of the sections. 9. If residue is left in the blade, wipe away large chunks of residue with the paint

brush and than spray 70% ethanol onto a Kimtech wipe a carefully wipe the blade and allow to air dry.

A. Always wipe away from the from the edge of the blade to ensure safety. 10. Label each slide on the frosted end and place in slide box at -20° for storage until

further processing. Notes:

It is best to cut approximately 12 sections with the settings in order to keep consistency.

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7.8 Hematoxylin and Eosin Staining Materials:

Slide rack Transfer containers and apparatus VWR micro glass cover (VWR, Cat: 48366-067) Cytoseal 280 glue (Thermo Scientific, Cat #: 8311-4)

Procedure: Container # Reagent Time 1 1x Phosphate Buffered Saline 1 minute 2 Erlich’s Hematoxylin 5 minutes 3 Tap Water (change every time) 1 minute 4 Distilled Water (change every time) 1 minute 5 30% Ethanol 1 minute 6 50% Ethanol 1 minute 7 Eosin 3 minutes 8 70% Ethanol (alternate the contents of this Rinse x 2 and the next dish after each use) 9 90% Ethanol Rinse 10 95% Ethanol Rinse 11 100% Ethanol 2 minutes 12 100% Ethanol 5 minutes 13 Xylene Substitute 5 minutes 14 Xylene Substitute 5 minutes Notes:

Refresh the Xylene Substitute after every 200 slides (substitute can be left of evaporate in the fume hood).

Eosin and Hematoxylin should stain thousands of slides, although filtering the stains prior to use if they have been sitting for longer than two days is best to remove lumps.

Eosin and Hematoxylin may be poured down the sink once no longer needed.

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7.9 Morphological Scoring System - Frequency of occurrence (items with a very low frequency of occurrence

were discarded) - Physiological relevance (items strongly related to eccentric exercise-induced

muscle damage) - 3 pictures of each sample taken~120 fibres per picture

0—absence of damage 1—moderate damage 2—severe damage Muscle Fibre

- Abnormal morphology (small, angulated or rounded fibres, swollen appearance of fibre splitting)

o 0= <4 o 1= 4-7 fibres o 2 > fibres or an entire fascicle

- Necrotic/(re)degenerating fibres [presence of infiltrating inflammatory cells (myophagocytosis), fragmented sarcoplasm, dark staining (hypercontracted fibres) and pale staining (necrotic fibres)

- Regenerating fibres were represented by early stage small basophilic myotubes and later by bluish-stained myofibres with central nuclei

o 0= absent o 1= 1-2 fibres o 2= >2 fibres

Inflammatory State

- Endomysial infiltrationsmall, mononuclear cells found in the endomysium o 0= <6 cells o 1= one cluster or ≥ 6 cells o 2= > 1 cluster of an entire fascicle infiltrated

- Perimysial infiltrationsmall, mononuclear cells found within the perimysium

o 0 = ≤ 10 cells o 1= >10 cells o 2= >2 clusters of widely diffused

Interstitial compartment

- Endomysium distensionspace b/w individual muscle fibres o 0= tight space o 1= moderately distended o 2= completely distended

- Perimysium distentionspace b/w fascicles o 0= tight space o 1= moderately distended o 2= completely distended