Effect of Prolonged Mechanical Ventilation on Sepsis Induced

Diaphragm Dysfunction

By

Angela Stamiris

Department of Experimental Medicine

McGill University, Montreal

August 2013

A thesis submitted to McGill University in partial fulfillment of the

requirements of the degree of Master of Science

© Angela Stamiris, 2013

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TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………..5

RÉSUMÉ…………………………………………………………………………...…7

AKNOWLEDGEMENTS………………………………………………..………….9

LIST OF ABBREVIATIONS……………………………………………..……….10

SECTION 1 –LITERATURE REVIEW

1.1 Introduction……………………………………………………………………..13

1.2 Skeletal muscle ……...…………………………………………..……………...14

1.3 Sepsis…………………………………….……………………………..….…….15

1.3.1 Skeletal muscle dysfunction in sepsis………..……………………………....15

1.4 Potential causes of diaphragm dysfunction in sepsis….….……………….….16

1.4.1 Role of Cytokines …….…………………………………………….....17

1.4.2 Oxidative stress and free-radical generation in skeletal muscle…….…18

1.4.3 Proteolysis……….. ………….………………………………………...20

1.5 Mechanisms of skeletal muscle protein degradation…………………………20

1.5.1 Ubiquitin-proteasome pathway...............................................................20

1.5.2 Calpain pathway………………………………………………......…....22

1.5.3 Caspase pathway…………………………………………………….....23

1.5.4 Autophagy-lysosome pathway………………………………………....24

1.6 Macroautophagy overview……………………………………………………..25

1.6.1 Modes of autophagy……………………………………………………27

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1.6.2 Autophagy and skeletal muscle dysfunction…………………………...29

1.7 Mechanical ventilation………………………………………………………….31

1.8 Effects of mechanical ventilation in sepsis………………………………….....33

2.0 Aims of this study……………………………………………………………….35

SECTION 2 – MATERIALS AND METHODS………………………………….36

2.1 Materials………………………………………………………………………...36

2.2 Animal experiments………………………………………………………….…36

2.3 Diaphragm contractility………………………………………………………..38

2.4 Diaphragm fiber cross-sectional area…..……..………………………………39

2.5 Plasma and diaphragm cytokine measurements……………………………...39

2.6 Measurements of mRNA expression…………………………………………..40

2.7 Immunoblotting ………………………………………………………………...41

2.8 Detection of protein oxidation………………………………………………….42

2.9 Statistical analyses……………………………………………………................43

SECTION 3 – RESULTS…………………………………………………………..44

3.1 Diaphragm contractility ……..………………………………………………...44

3.2 Diaphragm cross-sectional areas…………………………...……………….…44

3.3 5 Plasma and diaphragm cytokines………………..…………………………..44

3.4 Activation of protein degradation in the diaphragm…………………………45

3.5 Regulators of protein synthesis and autophagy.……………………………...46

3.6 Oxidative stress……………………..…………………………………………..47

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SECTION 4 – DISCUSSION....................................................................................48

SECTION 5 – TABLES…………………………………………………………….58

SECTION 6 – REFERENCES……………………………………………….…….60

SECTION 7 – FIGURE LEGENDS…………………………………………….....87

SECTION 8 –FIGURES…………………………………………………………....91

5

ABSTRACT

Severe sepsis is a systemic inflammatory response to an infection that often

leads to respiratory failure which requires patients to be mechanically ventilated.

Mechanical ventilation (MV) also leads to atrophy and weakness what is termed

ventilatory induced diaphragm dysfunction (VIDD). Both these conditions are

associated with upregulation of proteolytic pathways such as the ubiquitin proteasome

pathway and the autophagy-lysosomal pathway. However, the influence of combining

MV on sepsis-induced diaphragm dysfunction remains unknown. In this study, we

evaluate the influence of prolonged MV on sepsis-induced diaphragm dysfunction.

We studied four groups of rats. Group 1 animals were spontaneously

breathing and served as controls. Group 2 (LPS) animals received intraperitoneal

injection of E. coli lipopolysaccharide (LPS) and served as the sepsis group. Group 3

animals were mechanically ventilated for 12h. Group 4 animals received LPS

injection first and were then mechanically ventilated for 12h. Diaphragm contractility

was measured in-vitro and diaphragm fiber type atrophy was evaluated by measuring

fiber cross sectional areas. Activation of the proteasome, calpains, caspase-3 and

autophagy proteolytic pathways were evaluated using specific assays. Injection of

LPS and MV for 12 resulted in significant attenuation of diaphragm contractility and

the development of fiber atrophy. Combining MV with LPS administration resulted

in additional decline in muscle contractile performance but not additional atrophy.

Proteasome, calpain, caspase-3 and the autophagy proteolytic pathways were

activated in the LPS and MV groups and the combination of prolonged MV with

sepsis resulted in the potentiation of autophagy pathway but not proteasome, calpain

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and capase-3 activation. The AKT and mTORC1 pathways (inhibitors of proteolytic

pathways and activators of protein synthesis) were activated in response to LPS

administration but not by prolonged MV. Combining sepsis with prolonged MV

resulted in attenuation of AKT and mTORC1 activation compared to sepsis alone.

Interestingly, the AMPK pathway (activator of autophagy) is inhibited in response to

LPS administration and prolonged MV. Combining sepsis with prolonged MV

resulted in a milder degree of AMPK inhibition compared to LPS administration

alone. Finally, oxidative stress develops in response to LPS administration and

prolonged MV. Combining MV and sepsis resulted in worsening of oxidative stress.

These results indicate that prolonged MV worsens sepsis-induced diaphragm

contractile dysfunction and this worsening of function may be mediated by substantial

induction of autophagy and the development of severe oxidative stress.

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RÉSUMÉ

Le sepsis sévère est une réponse inflammatoire systémique consécutive à une

infection, pouvant conduire à la détresse respiratoire nécessitant le recours à la

ventilation mécanique.

La ventilation mécanique (MV) est responsable de l’atrophie et de la faiblesse

du diaphragme connue sous le nom de dysfonction diaphragmatique induite par la

ventilation (DDIV).

Ces deux mécanismes étiopathogéniques sont associés à l’activation de

plusieurs voies protéolytiques comme la voie du protéasome et la voie de

l’autophagie médiée par les lysosomes. Cependant, l’effet combiné de la ventilation

mécanique associée au sepsis n’est pas encore connu. Dans cette étude nous avons

évalué l’influence d’une ventilation mécanique prolongée sur la dysfonction

diaphragmatique induite par le sepsis.

Nous avons étudié quatre groupes de rats : le groupe 1 représentait le groupe

contrôle (animaux en ventilation spontanée) ; le groupe 2 (LPS) représentait le groupe

« sepsis » dans lequel les animaux recevaient une injection intrapéritonéale de

lipopolyssaccharide (LPS) d’E. Coli ; dans le groupe 3, les animaux étaient ventilés

pendant 12h et dans le groupe 4 les animaux recevaient d’abord l’injection de LPS

avant d’être ventilés pendant 12h.

La contractilité diaphragmatique était mesurée in vitro et l’atrophie musculaire

était évaluée en mesurant la surface de section des fibres. L’activation du protéasome

et des autres voies protéolytiques (calpaines, caspase 3 et autophagie) ont été étudiées

par tests spécifiques.

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L’injection de LPS et la ventilation mécanique entraînait une diminution

significative de la contractilité diaphragmatique et le développement d’une atrophie

des fibres musculaires. La combinaison de la VM à l’injection de LPS montrait une

altération plus importante de la contractilité diaphragmatique mais pas d’atrophie

supplémentaire.

Les différentes voies protéolytiques (protéasome, calpain, caspase-3 et

autophagie) étaient activées dans les groupes VM et LPS alors que la combinaison

des deux résultait en une potentialisation de l’autophagie mais pas de l’activation du

protéasome, des calpaines et de caspase 3. Les voies AKT et MTORC1 (inhibitrice de

la protéolyse et activatrice de la synthèse protéique) étaient activées en réponse à

l’injection de LPS mais pas par la VM prolongée. La combinaison du sepsis et de la

VM entraînait une atténuation de l’activation des deux voies AKT et MTORC1 en

comparaison au sepsis seul.

Par ailleurs, la voie AMPK (activatrice de l’autophagie) était inhibée en réponse à

l’injection de LPS et à la VM alors que la combinaison des deux entraînait seulement

une inhibition modérée de l’AMPK en comparaison à l’injection de LPS seule.

Enfin l’injection de LPS et la VM entraînait un stress oxydatif d’autant plus important

quand on combinait les deux facteurs.

Ces résultats montrent que la VM prolongée aggrave la dysfonction

diaphragmatique induite par le sepsis et que cette aggravation est due en partie à

l’activation de l’autophagie et au développement d’un stress oxydatif sévère.

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ACKNOWLEDGEMENTS

I would like to take the opportunity to thank Dr. Sabah Hussain for giving me

the chance to partake in an interesting project and explore an unfamiliar field of

study.I also appreciate your patience and guidance throughout this past year. I would

also like to thank Christine Mutter for her help and support throughout my master’s

degree. A big thank you goes out to all my colleagues, Mary Guo, Mashrur Rahman,

Sharon Harel, Raquel Ecchavaria, Marija Vujovic and Bernard Nkengfac. This

experience would not have been the same without you. Mash and Mary, you have

helped me tremendously this year. I am lucky to have met such great people and

developed friendships with you. Sharon, you are like a big sister to me and your

guidance was invaluable throughout my degree, thank you.

A well-deserved thank you goes to Dominique Mayaki for all your patience

and help. I would not have been able to complete this project without you.

Thank you to my thesis committee members, Dr. Simon Wing, Dr. Suhad Ali,

and Dr. Thomas Jagoe for the guidance you provided for my project.

A special thank you goes to all my friends and family outside the lab that had

to endure this phase as a graduate student.

Thank you to Dr. Celine Guichon. You are a great inspiration and I am very

glad I had the opportunity to share my chocolate with you.

Finally, I would like to thank Flora Golyardi for her support throughout this

past year. Without you, I would not have made it to where I am now. I truly have

made a great friend and I hope we maintain the friendship we have formed.

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LIST OF ABBREVIATIONS

AKT- Protein Kinase B

AMP- Adenosine monophosphate

AMPK- 5’-AMP-activated protein kinase

ATG- Autophagy-related gene

ALP- Autophagy-lysosome pathway

ATP- Adenosine triphosphate

ATPase- Adenosine triphosphatase

Atrogin-1 – muscle atrophy F-box

BNIP3- Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3

Ca2+

- Calcium

CASPASE- Cysteine-dependent aspartate-directed proteases

CMV- Controlled mechanical ventilation

CLP- Cecal ligation and perforation

CMA- Chaperone-mediated autophagy

COPD- Chronic obstructive pulmonary disorder

CSA- Cross-sectional area

DNHP- 2,4-dinitrophenylhydrazine

ER- Endoplasmic Reticulum

FIP200- Focal adhesion kinase (FAK) family interacting protein of 200 kDa

FOXO- Forkhead box protein O

FRC- Functional residual capacity

GABARAPL1- Gamma-aminobutyric acid receptor-associated protein-like 1

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HNE- 4-hydroxy-2-nonenal

HSP70- Heat shock protein 70

ICU- Intensive care unit

IGF-1- Insulin growth factor 1

IL-1- Interleukin-1

IL-6- Interleukin-6

i.p.- Intraperitoneal

LAMP2A- Lysosome-associated membrane protein type 2A

LC3- Microtubule-associated protein 1 light chain 3

LPS – Lipopolysaccharide

MHC- Myosin heavy chain

mTOR – Mammalian target of rapamycin

mTORC1- Mammalian target of rapamycin complex 1

MuRF-1 – Muscle ring finger 1

MV- Mechanical ventilation

NADPH- Nicotinamide adenine dinucleotide phosphate

N-acelyl-Asp-Glu-Val-Asp-Al- DEVD-CHO

NFB -Nuclear factor kappa-light-chain-enhancer of activated B cells

NO- Nitric oxide

OD- Optical density

p62- SQSTM1

p70S6K- p70 ribosomal protein S6 kinase

PCR- Polymerase chain reaction

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PI3KC3 – Class-III phosphatidylinositol-3-kinase

PINK1- PTEN-induced putative kinase 1

PTP- Permeability transition pore

PVDF- Polyvinylidene difluoride

ROS- Reactive oxygen species

RUNX1- Runt-related transcription factor 1

SDS-PAGE- Sodium dodecyl sulfate – polyacrylamide gel electrophoresis

SIRS- Systemic inflammatory response system

SOD1-Superoxide dismutase 1

SOD2- Superoxide dismutase 2

TNF - Tumor necrosis factor

TNF - Tumor necrosis factor alpha

ULK1- Uncoordinated-51-like kinase 1

ULK2- Uncoordinated-51-like kinase 2

UPP- Ubiquitin proteasome pathway

VIDD- Ventilatory-induced diaphragm dysfunction

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SECTION 1 –LITERATURE REVIEW

1.1 Introduction

Severe sepsis is a systemic inflammatory response to an infection -often

caused by endotoxin-producing gram negative bacteria-associated with organ

dysfunction that may lead to respiratory failure requiring patients to be mechanically

ventilated (71). Furthermore, patients who are mechanically ventilated for prolonged

periods of time are susceptible to respiratory muscle atrophy and weakness (60).

Atrophy and decreased contractility have been documented in both animal models of

mechanical ventilation (MV) (49, 98) and humans who are mechanically ventilated.

Increased proteolysis, decreased protein synthesis and recently, an increase in

autophagy have been shown to contribute to the diaphragm dysfunction and atrophy

occuring in the mechanically ventilated diaphragm (4, 75, 114).

It is reported that 70-100% of all septic patients have prolonged weakness

which will result in difficulty weaning from the ventilator (6). In fact, when compared

to other patients in the intensive care unit (ICU), septic patients were 2.4 times more

likely to require a longer weaning period due to a failed first attempt (6). Although it

has been shown that 4 h of MV prevents sarcolemmal injury caused by sepsis and

protects against force declines in the diaphragm (40), 12 h of MV have contrarily

shown significantly reduced muscle force generation and elevated levels of plasma

cytokines (33). These two models involve administering both the sepsis-inducing

endotoxin, lipopolysaccharide (LPS) and MV simultaneously however a more

clinically relevant model would be to administer MV post administration of LPS. This

14

thesis will thus examine the effect that MV has on the already damaged septic

diaphragm.

1.2 Skeletal Muscle

Skeletal muscles are striated muscle tissues that are composed of a mixture of

myofibers bundled in the perimysium. Through a process known as differentiation,

immature cells called myoblasts or myosatellite cells fuse together to form myofibers.

These myofibers contain myofilament proteins which are organized in myofibrils.

Together these form the sarcoplasm in which other organelles such as the

mitochondria, the sarcoplasmic reticulum which is the storage site for the calcium

(Ca2+)

ions used for contraction, and other cellular organelles are found (73).

Skeletal muscle fibers are classified as slow-twitch fibers, otherwise known as

type I, or fast twitch fibers which can be broken down into type IIa, IIb and IIx. Slow-

twitch skeletal muscle fibers have high levels of myoglobin and an abundance of

mitochondria. By comparison, fast-twitch skeletal muscle fibers have relatively low

levels of myoglobin and mitochondria and are thus classified as a glycolytic muscle

type. Unlike slow twitch fibers, when contracting, fast-twitch fibers generate

relatively higher force however, fast-twitch fibers are easily fatigued. Type IIa fibers,

also known as fast-twitch oxidative glycolytic fibers, are rich in mitochondria and

capillaries, but are also high in glycolytic enzymes making them both fast and

fatigue-resistant. Type IIx are the fastest but most easily fatigable fibers found in

humans and finally, type IIb are the fastest and most glycolytic fibers found in rodents

and are not expressed in humans (81).

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1.3 Sepsis

Sepsis is a medical term used to define a generalized host response to an infection

in the blood, urinary tract, lungs, skin, or other tissues caused by fungi, viruses,

parasites but most commonly by endotoxin-producing gram negative bacteria (19).

For example, LPS which is a major component of the outer membrane of gram-

negative bacteria is an endotoxin which induces a strong immune response. Infection

by such agents triggers an inflammatory cascade which may then lead to severe sepsis

followed by septic shock (19). Severe sepsis is defined as sepsis exacerbated by at

least one organ dysfunction, or by shock. Septic shock is defined as a sustained

decrease in arterial pressure which persists despite administration of adequate fluids

to the patient and requires further intervention such as intravenous vasopressor

medications (76). Severe sepsis has been reported to be increasing in incidence and

entails a mortality rate of at least 20% in most studies (97). Respiratory failure is a

frequent occurrence in patients with severe sepsis and has been shown to be a major

contributor of the high mortality with his condition (88).

1.3.1 Skeletal Muscle Dysfunction in Sepsis

One common finding in patients with sepsis is that they develop respiratory

muscle weakness. The damage to the diaphragm begins within hours and rapidly

increases over time. This means that a large number of septic patients will find

themselves mechanically ventilated for prolonged periods of time. Successful

weaning from the ventilator is largely dependent upon the diaphragms strength and

16

endurance following weaning. Unfortunately, 70-100% of patients report prolonged

weakness in both limb and respiratory muscles which ultimately results in respiratory

failure. The rectus abdominus muscle of septic patients, for example, is known to be

capable of producing less force than that of control patients. Although this is one of

few studies that have been done on humans, many have been done using animal

models. In mice for instance, it is consistently documented that the force of the

diaphragm decreases with sepsis compared to control mice.

Among the dysfunction in the septic diaphragm is sarcolemmal damage. By

injecting a tracer dye unable to permeate intact membranes known as Procion Orange

into the myofibers, Lin et al. showed that two animal models of sepsis, LPS injection

and cecal ligation and perforation (CLP), caused an increase in sarcolemmal damage

compared to controls. Furthermore, data also exist showing disturbances in Ca2+

homeostasis in septic skeletal muscle which is an important finding considering Ca2+

levels greatly affect regulation of muscle protein breakdown in sepsis (40).

1.4 Potential Causes of Diaphragm Dysfunction in Sepsis

The mechanisms responsible for sepsis-mediated dysfunction in skeletal

muscle are many and complex. Excess production of cytokines, excess free radical

generation, enhanced proteolytic activity; decreased protein synthesis and autophagy

have been identified as potential players in the induction of sepsis-induced muscle

injury.

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1.4.1 Role of Cytokines

Systemic inflammation is the first and major symptom of sepsis which leads

to the initiation of multiple organ failure. In early stages of sepsis, including the

systemic inflammatory response system (SIRS), the circulating levels of cytokines are

elevated and correlate strongly to the outcome of the patient. Although this release of

cytokines is a normal host response to infection, it initiates a secondary response

which intensifies the dysfunction and damage occurring in the tissue (33). For

example, in human skeletal cell cultures, pro-inflammatory cytokines such as

interleukin-1 (IL-1) and interleukin-6 (IL-6) are found to be expressed at low levels

but when these cells are exposed to high levels of exogenous cytokines, they begin to

upregulate and increase the level of pro-inflammatory cytokines (13; 90). The

diaphragm seems to be a particularly sensitive muscle in which there is an

exaggerated release of pro-inflammatory cytokines in comparison to limb muscles

(33).

Using a murine model of colon cancer, Acharyya et al showed that levels of

muscle wasting were dramatically increased when IL-6 was over-expressed (2).

Similar results have been documented in vitro, for example, when isolated muscle (8)

or muscle cell lines were exposed to tumor necrosis factor (TNF) or a combination of

cytokines for several h (78; 103; 143). TNF reduced muscle-specific force without

changing the muscle mass or the size of the skeletal muscle bundles and the cytokines

reduced the size of the cells and the amount of protein (78).

18

Although these studies were not directly involving the diaphragm, the data

from both the in vivo and in vitro studies suggest that pro-inflammatory cytokines are

involved in skeletal muscle weakness and wasting (2; 52; 89; 104).

1.4.2 Oxidative Stress and Free-Radical Generation in Skeletal

Muscle

Oxidative stress results from redox imbalance and is likely caused by

increased production of free radicals and a decline in endogenous antioxidant buffer

systems (39). Free radicals are any molecules that have an odd number of electrons.

They are highly reactive species that at low levels are important mediators for

signaling processes such as regulation of vascular tone, monitoring of oxygen tension

in the control of ventilation and erythropoietin production (39). Nitric Oxide (NO)

and reactive oxygen species (ROS), two types of free radicals, are typically generated

in these cases by tightly regulated enzymes such as nitric oxide synthase (NOS),

sarcolemmal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase

isoforms, and the mitochondrial electron transport chain respectively (39). When

generated in excess, these free radicals may damage the protein through modifications

that alter their function and may render them more susceptible to proteolytic attack

(39).

In skeletal muscle specifically, free radicals have been linked to alterations in

muscle performance under a variety of conditions (99; 106; 118; 127). Particularly in

sepsis, the aforementioned cytokines have been linked to the production of free

radicals in skeletal muscle. Indicators of oxidative stress caused by free radicals

19

include detection of protein carbonyls and 4-hydroxy-2-nonenal (HNE) by

immunoblotting. These markers have been shown to be increased in skeletal muscle

in sepsis (11; 59).

ROS seems to be modulating dysfunction in the diaphragm by affecting many

of the subcellular sites (120). These free radicals have been linked to a decrease in

myofilament function and muscle contractility causing muscle contractile dysfunction

(10). It has been shown that antioxidants such as catalase and N-acetylcysteine

actually prevent declines in muscle-specific force generation in animal models of

sepsis suggesting that these sepsis-induced dysfunctions of the skeletal muscle are

mediated by ROS (46; 47; 117; 120). For example, hamsters were injected with LPS

over 48h and four other groups of hamsters were made septic but were also

administered doses of various antioxidants every 12h. Authors showed that LPS

endotoxin reduced the diaphragmatic contractility compared to the control group and

they also showed that, in all cases, the antioxidants had a protective effect again the

dysfunction suggesting that ROS production plays a role in the dysfunction (120).

It is also known that proteolysis is increased in septic muscle in animals (126;

132). Although there is yet to be a concrete link between increased oxidative stress

and induction of the ubiquitin-proteasome pathway (UPP), it is known that oxidation

of proteins increases the susceptibility to degradation through the proteasomal

pathway (126). There is therefore high probability that oxidative stress and free

radicals also mediate the increase in proteolysis occuring in sepsis (126).

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1.4.3 Proteolysis

It is now widely accepted that muscle degradation occurs in a variety of

conditions such as starvation, immobilization, cancer and sepsis (1; 54). Many studies

have shown that under such circumstances components of the proteasome degradation

system are up-regulated such as the E3 ligases MAFbx (Atrogin-1) and muscle ring

finger 1 (MurF-1) and the 20s alpha subunit (1; 41; 54; 68; 100). The protein

degradation occuring in sepsis however cannot be fully attributed to the proteasomal

pathway and as such, a variety of systems for protein degradation are discussed

below.

1.5 Mechanisms of Skeletal Muscle Protein Degradation

There are four main proteolytic pathways that work in tandem to degrade

protein in skeletal muscles. These include the UPP, the calpain pathway, the caspase

pathway, and the autophagy-lysosome pathway (ALP).

1.5.1 Ubiquitin-Proteasome Pathway

The UPP has long been considered to be the primary system responsible for

muscle myofibrillar protein degradation (137). It is a quite complex, adenosine

triphosphate (ATP)-dependent mechanism involving a series of enzymatic reactions

in which proteins are targeted for degradation. This pathway involves a two-step

process consisting first of substrate recognition consisting of a conjugating cascade

followed by protein degradation through the 26s proteasome. The initial step requires

the activation of ubiquitin by E1 ubiquitin-activating proteins. Activated ubiquitin is

21

then transferred to E2 ubiquitin-conjugating enzymes which with E3 ligating

enzymes, which catalyzes the transfer of the active ubiquitin to a lysine residue on the

substrate protein. This process repeats itself forming a chain of ubiquitin molecules

attached to the targeted protein which signals to the proteasome that the protein is

ready to be degraded. The 26S proteasome consists of a 20S catalytic core and two

19S regulatory caps with three main catalytic activities: chymotrypsin-like, trypsin-

like, and caspase-like activities. The 19S caps serve to identify the substrates and

translocate them to the 20S core for degradation. It also involves ATPase activity

which is used to open the 20S core and unfold the substrate in order for it to be

properly inserted. Overall, this macromolecular complex cleaves tagged proteins into

short oligopeptides, which then undergo further degradation by cytoplasmic

peptidases while the ubiquitin get recycled (145).

Many UPP genes are up-regulated in conditions of muscle wasting such as

denervation, immobilization, and fasting, but few have been identified to be

expressed solely in skeletal muscles (41). A transcript profile in fasting and

immobilization models of rodent muscle atrophy led to the discovery of two muscle

specific E3 ligases Atrogin-1 and MuRF1 (18; 50). The role of these two genes in

atrophy has been confirmed through knock out animals showing a reduction in

denervation induced muscle wasting when Atrogin-1 and MuRF1 are absent (18). In

basal conditions, the insulin-like growth factor 1 (IGF-1)/PI-3 kinase/protein kinase B

(AKT) complex, which is involved in cell survival and hypertrophy inhibits atrogin-1

through the phosphorylation of forkhead box protein (FOXO)1 and FOXO3 by AKT

(111). This forces the FOXO family, which is a family of transcription factors, to

22

remain in the cytoplasm, bound to its docking protein 14-3-3, preventing it from

entering the nucleus and transcribing genes involved in protein degradation such as

Atrogin-1 and MuRF-1 (111). In catabolic conditions such as sepsis, IGF-1 and

insulin are low and there is a resistance to them leading to de-phosphorylation of

AKT thus de-phosphorylation of FOXO (111). FOXO can then move to the nucleus

where it transcribes genes promoting muscle wasting (111). Nuclear factor kappa-

light-chain-enhancer of activated B cells (NFB) transcription factor also plays a role

in increasing transcription of MuRF-1 (23). Whereas Atrogin-1 preferentially targets

MyoD, MuRF-1 targets myosin heavy chain (MHC) and potentially mediates titin

signaling (26).

In skeletal muscles, several stimuli are likely to activate the proteasomal

pathways but one stimulus that is repeatedly documented to do so is enhanced levels

of ROS. Exposure to ROS triggers significant increases in proteasomal activity and

enhanced expressions of MURF1 and Atrogin-1 in cultured skeletal muscle cells (77).

Other stimuli that have been shown to enhance proteolytic activity and up-regulation

of the muscle specific E3 ligases are the pro-inflammatory cytokine TNF and the

nuclear factor kappa-light-chain-enhancer of activated B cells (NFB) transcription

factor in culture skeletal muscle cells and in vivo muscles (23). Conditions such as

hypoxia have also been shown to be a strong activator of 20S activity and Atrogin-1

expression in cultured skeletal muscle cells (25).

1.5.2 Calpain Pathway

Although the proteasomal pathway is the major degradation pathway for

skeletal muscle it cannot degrade large myofilament proteins without the aid of

23

calpains. Calpains are Ca2+

-sensitive cysteine proteases, which become activated

upon spikes of Ca2+

and cleave proteins at selective sites (119; 126). In skeletal

muscles, the two isoforms that are present are μ-calpain and m-calpain as well as

calpain 3 (119). Their main function involves cleaving proteins, including

cytoskeletal elements involved in anchoring contractile element, which trigger the

release of smaller protein fragments that are ready to be degraded by the proteasome

(119; 126). Under normal circumstances, calpains under normal circumstances are

highly regulated and inactive. Excessive calpain activation can lead to unregulated

proteolysis causing tissue damage (14).

One model of sepsis used in animals is CLP. This model consists of

perforating the cecum which allows the release of fecal matter into the peritoneal

cavity which generates an exacerbated immune response induced by infection.

Calpains have been shown to be increased in rats undergoing CLP (16). Another

study confirmed that the calpain activity was increased when rats were mechanically

ventilated for 18 h (114).

1.5.3 Caspase Pathway

The caspase pathway is yet another important molecular pathway that has

been extensively described in terms of its contribution to apoptotic cell death. It also

seems to contribute to protein degradation in skeletal muscle fibers. Exercise-induced

oxidative stress and muscular dystrophy are examples of pathologies involving

skeletal muscle in which caspase activity has been shown to be enhanced (57). Few

studies have examined the effect of caspase in respiratory muscles of septic patients.

24

It has however been shown that administration of endotoxin resulted in a significant

increase in caspase 3 activation in the diaphragm (127; 128). Authors also found that

when they administered caspase inhibitor N-acelyl-Asp-Glu-Val-Asp-Al (DEVD-

CHO),diaphragm weakness was alleviated (128). Despite these findings, the exact

effects and mechanism of action of caspase-3 in the in-vivo model remain largely

unknown.

1.5.4 Autophagy-Lysosome Pathway

Another pathway that contributes to skeletal muscle protein degradation is the

ALP. Autophagy, which is the Greek word for self-cannibalization, is pathway that

has been evolutionarily conserved and is thus found in all eukaryotic organisms (86).

It is the primary mechanism by which long-lived proteins and organelles are degraded

in cells (86). The level of activity is low at basal level as it contributes to maintaining

homeostasis but can be rapidly upregulated during times of stress, starvation or

development (86). There are three types of autophagy that have been described in

mammalian cells: microautophagy, chaperone-mediated autophagy (CMA), and

macroautophagy (109).

Microautophagy involves the lysosome invaginating to take up areas of the

cytosol (110). Once inside the lumen, the content is degraded by hydrolases (110).

This is the class of autophagy that is the least studied and, therefore, no information is

yet available regarding mechanisms, regulation, or physiological significance of

microautophagy in skeletal muscle cells.

25

In CMA, substrates are comprised of soluble proteins that are recognized by a

complex that is constituted mainly by heat shock protein 70 (HSP70) and lysosome-

associated membrane protein type 2A (LAMP2A) protein (65; 70). This process is

very selective, in which only damaged proteins expressing amino acid sequence

KFERQ are recognized by the HSP70-LAMP2A complex (61). Once the protein has

been identified, it undergoes unfolding and is translocated to the lysosome lumen

where it is quickly destroyed (61). Under normal conditions, CMA activity is

relatively low; however, it is significantly induced in response to starvation or amino

acid deprivation (15). CMA is also strongly activated by oxidative stress in rat liver

and cultured mouse fibroblasts (66). Very little is known in terms of the level of

involvement, if any, of CMA in protein degradation of skeletal muscle. However a

study done on fasted rats has shown that CMA increased in the heart and liver but

was not found to increase in skeletal muscle (144).

1.6 Macroautophagy Overview

Macroautophagy, which is often simply referred to as autophagy, is the main

autophagy pathway that exists in skeletal muscles. Broadly, it is the cellular catabolic

process in which damaged organelles, protein aggregates, cytoplasm and oxidized

lipids are brought to the lysosomes for degradation (101). The main characteristic of

this form of autophagy is the formation of double-membrane vesicles known as

autophagosomes (101). In mammals, autophagy occurs under basal conditions but is

drastically increased under conditions of stress, starvation, remodeling, denervation

and other pathologies (101). Autophagy plays an important role in maintaining

26

cellular and tissue homeostasis in a mainly protective fashion (28). It is a complex

process which requires a series of tightly regulated steps involving the recruitment of

autophagy-related genes (ATG) (109). These genes were first identified in yeast but

have orthologs in higher eukaryotes which are classified according to their function at

different stages of the autophagy pathway (109).

The initiation of autophagy is dependent on complex 1 of mammalian target

of rapamycin (mTORC1), a kinase activated by AKT and suppressed by AMP-

activated protein kinase (AMPK) (109). Uncoordinated-51-like kinase 1 (ULK1)

protein is an important regulator of autophagosome formation which acts downstream

of mTOR. It exists in a complex with FIP200, ATG13 and ATG101 (63). This

complex is under the control of the AMPK and mTOR pathways (63). Under normal

conditions when nutrients are adequate, mTOR phosphorylates ULK1 on several

serine residues leading to reduction of ULK1 activity and inhibition of autophagy

(63). When the cell is deprived of nutrients of is starved, the metabolic sensor AMPK,

senses the decline in ATP levels and the increase in AMP levels (63). Consequently,

AMPK phosphorylates ULK1 on different serine residues than those targeted by

mTOR and this phosphorylation results in activation of ULK1 (67). ULK1 can then

phosphorylate ATG13 and initiation of autophagy is complete. The BECLIN1

complex which encompasses BECLIN1, PI-3 kinase CIII (PI3KC3), VIPS15,

UVRAG, and ATG14L is the complex necessary for the next step in autophagosome

formation which is autophagosome nucleation. Under normal conditions, BECLIN1

is anchored at the endoplasmic reticulum (ER) by BCl2 protein. When autophagy is

initiated, BCl2 is phosphorylated and thus dissociates from BECLIN1 allowing for the

formation of the BECLIN1 complex (95). Lastly, PI3KC3 is responsible for

27

generating phosphatidyl-inositol-3-phosphate (PI3P) which recruits other ATG

proteins required for the autophagosome formation (36; 146).

Autophagosome membrane elongation resembles closely the ubiquitin

conjugation system. ATG12 is first activated by ATG7 which acts like an E1 enzyme.

ATG12 is then transferred to ATG10, an E2-like enzyme and together associate with

ATG5 (130)(82). This complex then bind to ATG16L1 resulting in a large complex

that is involved in the formation of pre-autophagosomal structures (85). In the second

ubiquitin-like conjugation pathway, this complex acts like an E3 ligase and is

involved in the lipidation of microtubule-associated light chain-3 (LC3). First, LC3

undergoes cleavage by a cysteine protease, ATG4 into the cytosolic form of LC3-I.

ATG7, ATG3, another E2-like molecule, and the ATG16L1 complex conjugate

phosphatidylethanolamine (PE) to LC3-I resulting in the generation of LC3-II protein.

This is the lipidated form of LC3-I which is incorporated into newly forming

autophagosome membranes (131). LC3-II remains incorporated in the

autophagosomal membranes and is, therefore, degraded upon fusion with the

lysosome. For that reason it is commonly used as a marker of autophagosome

formation (64). The outer membrane of the mature autophagosome can now fuse with

the lysosome releasing the proteins and organelles for degradation by proteases,

lipases, nucleases and hydrolases (146).

1.6.1 Modes of Autophagy

It was initially believed that autophagy was a non-selective process in which

cargo was randomly targeted; however recent evidence shows that organelles such as

28

the ER, peroxisomes, mitochondria and even lipid globules are selectively targeted

for degradation by ER-phagy, pexophagy, mitophagy and macrolipophagy

respectively (101; 142). Mitochondria have been studied more closely because, as

they are the major source of ROS, they are more prone to ROS damage. Although

mitochondria are the main source of ATP required for muscle contraction, when they

are damaged they can initiate several pathways that trigger apoptotic cell death (139).

Getting rid of these damaged mitochondria is therefore important for the well-being

of cells (83). When the mitochondria are depolarized due to stress and damage, the

mitophagy pathway is activated (51). The exact signaling mechanisms involved in

targeting of the dysfunctional mitochondria by autophagosomes remain under

investigation (101).

It has been suggested that a potential mechanism responsible is the PINK-

PARKIN axis. Under normal conditions, mitochondrial serine/threonine kinase

PTEN-induced putative kinase 1 (PINK1) undergoes continual degradation by

mitochondrial proteases. When a mitochondrion is depolarized, PINK1 starts to be

expressed on the outer mitochondrial membrane and recruits PARKIN which is an E3

ligase (116). PARKIN then helps the autophagosome engulf the organelle by

triggering the recruitment of the adaptor protein SQSTM1 (p62) to the outer

membrane followed by the binding of this protein to LC3B protein located on the

inner membrane of forming autophagosomes (94). Although it has been confirmed in

cultured cells, the functional significance of this axis in mitophagy under

physiological or pathological conditions in vivo has yet to be determined.

Another important signaling cascade is required for removal of mitochondria

under conditions unrelated to changes in permeability transition pore (PTP). BNIP3

29

and BMIP3L are two proteins recruited to dysfunctional mitochondria under

conditions such as starvation, fasting and denervation. The binding of

BNIP3/BNIP3L to LC3IIB has been demonstrated in cultured cells thus initiating the

selective removal of mitochondria by autophagy. Lastly, mitochondrial fission

machinery is actively involved in muscle wasting in mice. This fission is a selective

stimulus for autophagy recruitment and when inhibited, muscle loss is prevented

during denervation. Taken together, this indicates that mitophagy is a necessary

mechanism to maintain cell homeostasis.

1.6.2 Autophagy and Skeletal Muscle Dysfunction

Loss of muscle occurs in various conditions such as inactivity, denervation,

fasting and cancer (74). Although autophagy is of great interest of late, there is still

much to be understood about the functional importance of the protein degradation

pathway in proteolysis of muscle proteins and organelles. It is known now that basal

autophagy plays a critical protective role in maintaining muscle mass. This was

confirmed by the selective deletion of ATG7, an important enzyme involved in the

autophagy process in animal skeletal muscle. The knock-out mice showed abnormal

morphological features in skeletal muscles and their mitochondria. This result

therefore showed that suppression of autophagy triggers weakness and atrophy in

skeletal muscle (82). More specifically, there was a large increase in abnormal

mitochondria and oxidative stress, and there was an accumulation of protein

aggregates in the ATG7 knockout mice suggesting that autophagy plays a role in

preventing cell death in skeletal muscles (82).

30

Other models exist to demonstrate the role of autophagy in skeletal muscle.

One such model is denervation. In denervation-induced muscle atrophy there is a

reduction in mitochondrial content and function which perhaps can be explained by

increased mitophagy (3; 148). This decrease in muscle mitochondrial density is

indicative of increased autophagy in order to ensure the clearance of the damaged

organelle. Starvation is also a common stimulus that triggers muscle atrophy. Fasting

has been linked to an up-regulation of genes involved in autophagy signaling as well

as an increase in autophagosome number and LC3-II. FOXO1 and FOXO3 are

upregulated in starvation-induced skeletal muscle atrophy, which may explain this

elevated level of autophagosome formation (115). FOXO transcription factors are

found downstream of the IGF-1/insulin/AKT in the signaling pathway (133). FOXOs

regulate the expression of genes involved in apoptosis, cell growth and other cellular

processes. FOXO1 and FOXO3 in particular have been shown to be involved in the

transcription of Atrogin-1 and MuRF-1. When FOXOs are inactive they are

phosphorylated and anchored in the cytosol to protein 14-3-3. When AKT is

inhibited, FOXOs become dephosphorylated and can translocate to the nucleus (134)

to transcribe not only the muscle specific E3 ligases but also selective genes involved

in autophagy such as LC3, ATG7, BNIP3 and GABARAPL1 (80).

Sepsis-induced muscle atrophy also leads to augmented levels of autophagy.

When mice are infected with LPS, atrophy is induced. LPS-mediated severe sepsis

triggers morphological and functional abnormalities in mitochondria which lead to

increased mitochondrial injury, pathological opening of the PTP which is known to

increase ROS production, (22) and inhibition of mitochondrial biogenesis. As

mentioned earlier, it is suggested that damaged mitochondria are targeted by

31

autophagy which would therefore mean that in sepsis, autophagy would be

upregulated. In fact, there is a substantial increase in autophagy confirmed by the

increased lipidation of LC3-II and the enhanced expression of autophagy-related

proteins including BECLIN1, p62, PI3KC3, ATG5 and ATG12 (87).

Despite its pro-survival role, excess autophagy can be harmful to overall

muscle function. Evidence of these detrimental effects has been shown in animal

models of denervation. When RUNX1, a suppressor of autophagy particular to the

denervation model, is inactivated in mice muscle, these mice end up with

uncontrolled autophagy which exacerbates the atrophy caused by denervation

compared to wild-type mice (140). This demonstrates the importance of RUNX1 in

the maintenance of muscle mass. Similarly, when Jumpy, a suppressor of autophagy

was mutated in mice, autophagy was significantly over-induced and resulted in

centronuclear myopathy, a condition heavily involving muscle atrophy (138). This

evidence suggests that when suppressors of autophagy such as RUNX1 and Jumpy

are inhibited, there is a disturbance in muscle fiber characteristics resulting in

atrophy.

1.7 Mechanical Ventilation

Mechanical ventilation is an essential life-saving tool that is used in the ICU

as an intervention for respiratory failure. It functions as an artificial breathing

mechanism by inflating the lungs via positive pressure thereby sustaining alveolar

ventilation (32). Indications to intervene with MV include reduced respiratory drive,

chest wall abnormalities, respiratory muscle fatigue, inefficient gas exchange,

32

ventilation-perfusion mismatch, decreased functional residual capacity (FRC),

chronic obstructive pulmonary disorder (COPD), recovery from anaesthesia, drug

overdoses and neuromuscular diseases. Despite its life saving capabilities, difficulties

in weaning patients from the ventilator occur in 20–30% of patients (7). This number

becomes more elevated in patients with COPD, ranging from 35% to 67%. There is

accumulating evidence that difficulty weaning patients off MV is associated with

diaphragm inactivity causing diaphragm weakness what is termed ventilator-induced

diaphragm dysfunction (VIDD) (32; 49).

To date, 57 studies have used animal models to determine the effect of MV on

the diaphragm (32). These studies have shown that within a 6-24 h period, controlled

mechanical ventilation (CMV) leads to VIDD in healthy young animals. CMV has

also been shown to cause muscle atrophy as early as 18 h after introducing MV (62).

Protein degradation has been documented in animals subjected to 18 h of MV. These

studies have also identified that the three major pathways responsible for protein

degradation including the UPP, the caspase and calpain pathway and the ALP are all

upregulated (32). Shanely et al. for example, documented an increase in proteolysis

and atrophy in the mechanically ventilated diaphragm as indicated by the more than

doubling of the calpain-like activity and the 5 fold increase in the 20S proteasome

activity. They also showed an increase in diaphragmatic oxidative stress. This is

important, because oxidative stress can contribute to both muscle atrophy and

contractile dysfunction (114). It is well known that unloading of a muscle due to

immobilization is associated with oxidative stress (72). CMV is a clinical form of

unloading a muscle because the diaphragm is inactive. In fact, within 6 h of CMV, it

has been shown that there is oxidative stress in the diaphragm (149). Oxidative stress

33

may also activate proteolytic systems by increasing the levels of free cytosolic Ca2+

by decreasing Ca2+

ATPase activity which inhibits the removal of Ca2+

from the cell

and promotes intracellular Ca2+

accumulation (4). This accumulation will then

activate proteolytic systems dependent upon Ca2+

concentration such as the calpain

pathway thereby inducing muscle atrophy (4).

This animal model is not without limitations however, because it is technically

difficult to maintain animals on MV for longer than 24 h, while human patients are

often mechanically ventilated for extended periods of time. There are approximately

19 studies that have looked at the effect of MV on the human diaphragm in which

muscle fiber cross-sectional area (CSA), diaphragm pressure generation, gene

expression and cellular and molecular mechanisms of VIDD were examined (32).

Overall, the data found in these studies supports the findings in the animal studies.

For example, Levine et al. studied biopsies of brain-dead organ donors and compared

them to intraoperative biopsy specimens. Fibers in the diaphragm of the case subjects

were shown to be considerably smaller than those of the control subjects. Following

analysis of gene expression, the case subjects were found to have 3 times the

expression of Atrogin-1 mRNA and 6.9 times as much expression of MuRf-1 mRNA

transcripts which confirms there was indeed an increase in the UPP in the

mechanically ventilated diaphragms (75).

1.8 Effects of Mechanical Ventilation in Sepsis

As previously mentioned, patients with sepsis often require MV due to

respiratory failure. There are two hypotheses concerning the effects of MV on the

34

septic diaphragm. It has been suggested that MV helps protect the diaphragm from

sepsis-induced injury. This was found when two groups of rats were infused with

LPS, one of which was MV for 4 h post infusion. MV actually rescued the diaphragm

from the damage seen in the sepsis group alone. Upon injecting the muscles with a

tracer dye unable to permeate a healthy sarcolemma, it was also noted that the muscle

fluoresced less orange in the MV+LPS group than sepsis alone further confirming the

rescuing effects of MV (40). In a more realistic clinical setting, using critically ill

patients, respiratory muscle strength was measure by stimulating the phrenic nerve

and measuring transdiaphragmatic pressure. This study showed that those that were

MV had half the normal values of twitch Pdi. Contrary to the previous study, this one

suggests that long term MV hinders the contractility of the diaphragm in a critically

ill patient (141). This was confirmed in rats where it was shown that MV decreases

the contractility of the septic diaphragm in a time dependent manner (98).

It is evident from this literature review that CMV decreases diaphragm

strength in humans and animals. Despite data showing a short duration of MV (4 h)

protects against sepsis-induced damage, such a short period of MV is unrealistic. It

has been shown that MV and sepsis administered simultaneously also has deleterious

effects on the diaphragm however the role of MV on a diaphragm already damaged

by sepsis has yet to be studied.

35

2.0 Aims of this study

We hypothesize that both prolonged MV and sepsis will impair diaphragm function

in rats and that combination of MV and sepsis will lead to worsening of diaphragm

contractile dysfunction. Specifically, we postulate that prolonged MV and sepsis

will impair diaphragm contractility, activate proteolytic activities including the

proteasome and autophagy and will worsen the extent of oxidative stress in the

diaphragm. We also postulate that combining prolonged MV and sepsis will lead to

worsening of diaphragm contractile dysfunction, further activation of the

proteolytic processes and worsening of oxidative stress in the diaphragm.

36

SECTION 2 – MATERIALS AND METHODS

2.1 Materials: Antibodies for LC3B, BECN1, SQSTM1 (p62), PI3KCIII, ATG7,

phospho-AKT (Ser473

), AKT, phospho-AMPK (Thr172

), and AMPK, were

purchased from New England Biolabs (Whitby, ON). Antibody for BNIP3 was

purchased from Sigma-Aldrich (Oakville, ON) and -TUBULIN was obtained from

Developmental Studies Hybridoma Bank (Iowa, USA). Antibody for II

SPECTRIN was obtained from Enzo Life sciences. Antibody for PARKIN

(PARK8) was purchased from Thermo Scientific (Rockford, IL). Detection of

protein carbonylation was performed with OxyBlot Protein Oxidation Detection Kit

(Millipore Inc., Billerica, MA). Antibody for ubiquitin was obtained from Cell

Signaling Technology (Boston, MA). HNE antibody was purchased from R&D

Systems, (MN, USA). Antibodies for E2 ubiquitin-conjugating enzymes UBE2B

(UBC2) and UBE2D2 (UBC4) were gifts from Dr. S. Wing (McGill University).

2.2 Animal experiments: The study was approved by the animal experiments

committee of the Medical Faculty of the Katholieke Universiteit Leuven. Adult male

Wistar rats (350-500g) were examined. All animals were anesthetized with sodium

pentobarbital (60 mg/kg) and were tracheotomized and the right external jugular vein

and carotid artery were cannulated for the continuous infusion of anesthesia (sodium

pentobarbital 2 mg/100g/h) and heparin (2.8U/ml/h) using osmotic pumps (Pilot A2,

Fresenius, Schelle, Belgium). Body temperature was continuously measured and

maintained at 37ºC using a heated blanket. All animals were breathing humidified air

enriched with O2 and maintained at 37°C. The animals were divided into four groups.

Group 1 (control group, n=8) served as a control and animals in this group were left

37

to breath spontaneously and were euthanized 24h later. Group 2 (LPS group, n=10)

animals received an intraperitoneal (i.p.) injection of E. coli lipopolysaccharide (LPS,

5 mg/kg) and were left spontaneously breathing. Animals were euthanized 24h after

LPS injection. Group 3 (MV group, n=8) received i.p. of normal saline after 12h

were mechanically ventilated for 12h while group 4 (LPS+MV group, n=8) received

i.p. of E. coli LPS (5 mg/kg) and after 12h of the injection they were mechanically

ventilated for 12h. MV was achieved with a volume-driven small-animal ventilator

(Harvard Apparatus model 665A, Holliston, MA) (tidal volume, ± 0.5 ml/100 g;

frequency of breathing, 55–60 breath/min). To maintain fluid volume status,

endotoxin treated animals were given subcutaneous injections of saline, 60

ml/kg/12h. During the duration of mechanical ventilation, continuous care to the

animals was performed including expressing the bladder, lubricating the eyes,

rotating the animal and passive movement of the limbs. Arterial blood pressure was

monitored during the protocol and blood gases were measured at dissection time. To

maintain blood pressure in endotoxemic rats, intravenous infusion of norepinephrine

combined with Voluven was used when necessary. Upon the completion of the

experimental period (24h), the costal diaphragm was quickly removed through a

laparotomy, and immediately immersed in a cooled, curarized, oxygenated Krebs

solution containing (in mMol/L) : NaCl 137, KCl 4, CaCl2 2, MgCl2 1, KH2PO4 1,

NaHCO3 12, glucose 6.5. Two small rectangular bundles (width<2mm) from the

middle part of the lateral costal region of each hemidiaphragm were obtained by

careful dissection parallel to the long axis of the fibers. Another piece of the right

diaphragm was processed for cross sectional area measurements (see below) and the

38

rest of the diaphragm was frozen in liquid nitrogen and stored under -80ºC for further

analysis.

2.3 Diaphragm contractility: Both ends of each diaphragm bundle were tied with

silk sutures to serve as anchoring points. The bundles were suspended in a tissue bath

containing Krebs solution and continuously aerated with 95% O2 and 5% CO2.

Temperature was maintained at 37ºC using a thermostatically controlled water pump.

The bundles were placed in between two large platinum stimulating electrodes,

anchored at the bottom to a rigid support and at the top fastened to an isometric force

transducer (Maywood Ltd., Hampshire, U.K.) connected to a micrometer. Signals

were amplified and recorded on computer via analog to digital conversion (DT-

2801A) using Labdat (Labdat/Anadat, RHT-Infodat, Montreal, Canada). Stimulations

were delivered through a Harvard 50-5016 stimulator (Edenbridge, Kent, U.K.),

connected to a power amplifier made from power one mode HS24-4.8, developed by

computer technology resources centre, University of Virginia (R.J. Evans, 1983).

Optimal muscle length (Lo) for peak twitch force was established for each bundle.

The following measurements were performed at Lo, after a thermo-equilibration

period of 15 min:

a) Maximum twitch force was obtained from two successive twitch stimulations (1

Hz). The highest value was chosen.

b) Maximal tetanic force was obtained by stimulating diaphragm strips twice at 160

Hz (duration of 250 msec) with a two minute interval. Each pulse had a duration of

0.2 msec. Tetanic force was taken as the maximal tension elicited at 160 Hz.

39

c) The force-frequency relationship was measured, using the following order or

frequencies with two minutes of interval in between the stimulations: 25, 50, 80, 120

Hz.

At the end of the in vitro experiment, each muscle bundle was removed from

the bath and its length, width and thickness were measured at Lo. They were blotted

dry and weighed. All tensions were normalized for CSA.

2.4 Diaphragm fiber cross sectional areas: The right costal region of the diaphragm

was folded, cut transversely, and placed at excised length on a cork holder, with the

fibers oriented perpendicularly to the surface of the cork. The preparations were

frozen in isopentane cooled with liquid nitrogen. Afterwards, serial sections parallel

to the cork were cut at 10μm thickness with a cryostat kept at -20°C. Two sections of

each muscle were stained for routine H&E, whereas the other serial sections were

stained for adenosine triphosphatase (ATPase) after acid pre-incubation at pH 4.5 and

4.3. Based on their histochemical reactions, fibers were identified as slow-twitch type

I, fast-twitch type IIa or fast-twitch type IIx/bfibers. CSA were determined from the

number of pixels within the outlined borders using a Leitz Laborlux S. microscope

(Wetzlar, Germany) at x20 magnification, connected to a computerized image system

(Quantimet 500, Leica, Cambridge Ltd., U.K.). Around 150 fibers were used to

calculate CSA and proportions of all fiber types.

2.5 Plasma and diaphragm cytokine measurements: Interleukin-6 (IL-6),

interleukin 1 (IL-1) and tumor necrosis (TNF-) were the plasma and diaphragm

lysates using a custom SearchLight rat cytokine proteome array (Aushon

Biosystems). Diaphragm lysates as described below.

40

2.6 Measurements of mRNA expression: Total RNA was extracted from

diaphragm samples using a GenElute™

Mammalian Total RNA Miniprep Kit

(Sigma-Aldrich, Oakville, ON). Quantification and purity of total RNA was

assessed by A260/A280 absorption. Total RNA (2µg) was then reverse transcribed

using a Superscript II

Reverse Transcriptase Kit and random primers (Invitrogen

Canada, Inc., Burlington, ON). Reactions were incubated at 42°C for 50min and at

90°C for 5min. Real-time PCR detection of mRNA expression was performed using

a Prism®

7000 Sequence Detection System (Applied Biosystems, Foster City, CA).

Specific primers were designed to quantify expressions of rat Gabarapl1, Uvrag,

Ambra1, Atrogin-1, Murf-1, Nedd4, Sod1 (superoxide dismutase-1), Sod2

(superoxide dismutase-2), Catalase, Ulk1, Ulk2 and -Actin transcripts (Table 1).

We chose to detect Gabarapl1, Uvrag, Ambra1, Ulk1 and Ulk2 autophagy-related

genes because of their importance in the initial phase of autophagosome formation,

expansion of the isolation membrane Atrogin-1, Murf-1 and Nedd4 are muscle

specific E3 ligases involved in the proteasome pathway while Sod1, Sod2 and

catalase are antioxidant enzymes. The gene -Actin served as an endogenous

control transcript. One l of reverse-transcriptase reagent was added to 25µl of

SYBR Green® (Qiagen Inc, Valencia, CA) master mix and 3.5µl each of 10µM

primers. The thermal profile was as follows: 95°C for 10 min; 40 cycles each of

95°C for 15s; 57°C for 30s; and 72°C for 33s. All real-time PCR experiments were

performed in triplicate. A melt analysis for each PCR experiment was performed to

assess primer-dimer formation or contamination. For each target gene, cycle

41

threshold (CT) values were obtained. Relative mRNA level quantifications of

target genes were determined using the threshold cycle (ΔΔCT) method.

2.7 Immunoblotting: Frozen diaphragm samples were homogenized in

homogenization buffer (10mM tris-maleate, 3mM EDTA, 275mM sucrose, 0.1mM

DTT, 2g/ml leupeptin, 100g/ml PMSF, 2g/ml aprotinin, and 1mg/100 ml pepstatin

A, pH 7.2). Samples were centrifuged at 5000 rpm for 10min in the cold room. Pellets

were discarded and supernatants were designated as crude homogenate. Total muscle

protein levels in each sample were determined using the Bradford protein assay

technique. Crude homogenate samples (25-50 g/sample) were mixed with SDS

sample buffer, boiled for 5min at 95° C, then loaded onto tris-glycine sodium dodecyl

sulfate polyacrylamide gels (SDS-PAGE) and separated by electrophoresis. Proteins

were transferred by electrophoresis to polyvinylidene difluoride (PVDF) membranes

and blocked with 1% bovine serum albumin or milk for 1h at room temperature.

PVDF membranes were incubated overnight with primary antibodies at 4°C then

washed and incubated with horseradish peroxidase-conjugated secondary antibody.

Specific proteins were detected with an enhanced chemiluminescence kit (ECL,

Millipore, Billerica, MA). Equal loading of proteins was confirmed by stripping each

membrane and re-probing with anti-β-TUBULIN antibody. Blots were scanned with

an imaging densitometer and optical densities (OD) of protein bands were quantified

using Gel-Pro Analyzer software (MediaCybernetics Inc., Rockville MD). These

ODs were then normalized per -TUBULIN OD. Changes in LC3B-I and LC3B-II

protein levels were also expressed as LC3B-II/LC3B-I ratios. For detection of calpain

and caspase-3 activity, we used immunoblotting with -II SPECTRIN antibody. This

42

protein is a substrate for both calpains and capase-3 and that the cleavage product of

intact -II SPECTRIN by calpains gives bands at 150 and 145kDa and when cleaved

by capase-3 at 150 and 120kDa. Intact -II SPECTRIN is detected at 260 kDa. The

intensities of cleaved bands were normalized for intact SPECTRIN band per a given

sample.

2.8 Detection of protein oxidation: To evaluate the degree of protein oxidation

and carbonyl formation, HNE protein-adduct formation (index of lipid

peroxidation) was detected using immunoblotting techniques described above.

Total protein carbonyl levels were measured in muscle homogenates using an

OxyBlotTM Protein Oxidation Detection Kit (Millipore). Briefly, carbonyl groups on

protein side chains were derivatized to 2,4-dinitrophenylhydrazone by reaction with

2,4-dinitrophenylhydrazine (DNPH), according to manufacturer's instructions. In

brief, 8µg of protein were used per derivatization reaction. Proteins were denatured

by addition of 12% sodium dodecylsulfate (SDS). Samples were subsequently

derivatized by adding 10µl of 1x DNPH solution and incubated for 15min. Finally,

7.5µl of neutralization solution and 2-mercaptoethanol were added to the sample

mixture. DNP-derivatized proteins were loaded onto 12% tris-glycine SDS

polyacrylamide gels then separated by electrophoresis. Proteins were transferred by

electrophoresis to methanol pre-soaked PVDF membranes then blocked with 5%

non-fat dry milk for 1hr at room temperature. PVDF membranes were then

incubated overnight at 4°C with a polyclonal anti-DNP moiety antibody. PVDF

membranes were washed several times with buffer and incubated with horseradish

peroxidase (HRP)-conjugated anti-rabbit secondary antibodies for 1h. Specific

43

proteins were detected with a chemiluminescence (ECL) kit. Blots were scanned

with an imaging densitometer and OD of each lane was quantified using Gel-Pro

Analyzer software (MediaCybernetics Inc., Rockville MD).

2.9 Statistical analyses: Statistical analysis was performed using the SAS Statistical

package (SAS Institute, Cary, NC) and SigmaStat software. Normality was assessed

using the Shapiro-Wilk test. Comparisons between the four groups of animals were

performed using one-way analysis of variance, followed by a Tukey post hoc test.

When the distribution is not Gaussian, non-parametric testing was performed by a

Kruskall-Wallis test, followed by Dunn’s multiple comparison test. Spearman rank

coefficients were used to evaluate relationships between variables. A p-value <0.05

was considered as significant. Data are expressed as means ± standard errors of the

means.

44

SECTION 3- RESULTS

3.1 Diaphragm contractility: Figure 1 illustrates the effects of LPS injection, MV

and combination of LPS injection and MV on rat diaphragm contractile performance.

Maximum twitch force values in the LPS and MV groups were significantly lower

than the control values. In the LPS+MV group, maximum twitch force declined

further compared to the control values (Figure 1). Similarly, maximum tetanic force

values in the LPS and MV groups were significantly lower than control values

(Figure 1). In the LPS+MV group, maximum tetanic force declined further compared

to values measured in the LPS group (Figure 1). The force-frequency relationships in

the LPS and MV groups were shifted downward compared to the control relationship

and the combining LPS injection with MV resulted in further shift downward in the

diaphragm force-frequency relationship suggesting that the decline in diaphragm

force observed with LPS injection or MV was worsened when LPS injection was

combined with MV (Figure 1).

3.2 Diaphragm cross sectional areas: Significant decline in cross sectional areas of

type I and type IIx/b fibers were observed in the LPS, MV and LPS+MV groups

compared to the control group (spontaneously breathing rats)(Figure 2). No

significant differences in cross sectional areas of type IIa fibers were observed among

the four groups of animals.

3.3 Plasma and diaphragm cytokines: Plasma TNF values were not detectable in

all animals. Plasma IL-1 values in the LPS group were significantly higher than the

control values whereas plasma IL-1 values of the MV and LPS+MV groups were

not different from control values (Figure 3A). These results indicate that combining

45

LPS injection with LPS resulted in attenuation of LPS-induced rise in plasma IL-1.

Plasma IL-6 levels were not detected in the control animals (Figure 3B). Plasma IL-6

values in the LPS and MV groups were significantly induced and these values in the

LPS+MV group rose substantially higher than the LPS group suggesting that

combination of LPS+MV resulted in potentiation of plasma IL-6 levels (Figure 3B).

No differences among the four groups of animals were observed in terms of

diaphragm TNF levels (Figure 4A). Diaphragm IL-1 levels rose significantly in

the LPS, MV and LPS+MV groups compared with control values (Figure 4A).

Diaphragm IL-6 levels rose in the LPS and MV groups compared with the control

group (Figure 4B). Further rise in diaphragm IL-6 values were observed in the

LPS+MV compared with the LPS group alone (Figure 4B) suggesting that MV

potentiated LPS-induced rise in IL-6 levels.

3.4 Activation of protein degradation in the diaphragm: Measurements of protein

ubiquitin conjugation levels in the diaphragm revealed that these levels rose

significantly in the LPS and MV groups compared with control values (Figure 5). A

further increase in protein ubiquitin conjugation levels was observed in the LPS+MV

group (Figure 5).

Diaphragm protein levels of the protein conjugase UBC4 rose significantly in

the LPS, MV and LPS+MV groups compared to the control group (Figure 6A-B). No

significant differences in the levels of protein conjugase UBC2 and the subunits of

the 20S proteasome were observed in the four groups of animals (Figure 6A-B). The

expression of mRNA levels of muscle specific E ligases Atrogin-1 and Murf-1 rose

significantly in the LPS, MV and LPS+MV groups compared to control values

46

(Figure 6C). The expression of the E3 ligase Nedd4 rose significantly only in the

LPS+MV compared to the control group (Figure 6C). Figure 7 illustrates calpain and

capase-3 activities measured in the diaphragm of the four groups of animals. Calpain

activity rose significantly in the LPS and MV groups but not in the LPS+MV group

(Figure 7). Caspase-3 activity increased significantly higher than control values only

in the LPS group (Figure 7).

During autophagic vacuole formation, LC3B protein is cleaved and

conjugated to phosphatidylethanolamine to generate a fast-migrating form, LC3B-II

(55). Lc3B protein immunoblotting revealed a significant increase in LC3B-II and

LC3B-II/LC3B-I ratio in the diaphragm of LPS and MV groups compared to control

values (Figure 8). Further increases in LC3B-II and LC3B-II/LC3B-I ratio were

observed in the LPS+MV group compared to the LPS group alone (Figure 7).

mRNA and protein levels of several autophagy-related genes involved in

autophagosome formation (BECN1, Gabarapl1, Uvrag and Ulk1) and selective

targeting of mitochondria by autophagosomes (SQSTM1 and PARKIN) rose

significantly the diaphragm of the LPS and MV groups compared to control values

(Figure 9). A further increase in PARKIN, Gabarapl1, Ambra1 and Uvrag

expression was observed in the LPS+MV group compared to the LPS group (Figure

9).

3.5 Regulators of protein synthesis and autophagy: AKT phosphorylation on

Ser473

, but not total AKT protein, was significantly increased in the diaphragm of

the LPS group (Figure 10A-B). No significant alterations in AKT phosphorylation

or total AKT levels were observed in the MV and LPS+MV groups compared to the

control group (Figure 10A-B). To evaluate mTORC1 activity, phosphorylation of

47

P70S6K1 (serine/threonine kinase that phosphorylates the ribosomal protein S6)

was evaluated with immunoblotting. P70S6K1 phosphorylation in the LPS, MV

and LPS+MV groups was significantly greater than in the control group. P70S6K1

phosphorylation levels in the LPS+MV group was relatively lower than that of the

LPS group (Figure 10C-D). Phospho-AMPK levels significantly declined in the

diaphragm of the LPS, MV and LPS+MV groups compared to the control group

(Figure 10E-F). Phospho-AMPK levels in the LPS+MV group were relatively

higher than that of the LPS group (Figure 10E-F).

3.6 Oxidative stress: The development of oxidative stress in the diaphragm of the

LPS, MV and LPS+MV groups was indirectly assessed by measuring protein

carbonyl formation and 4HNE protein adduct formation as well as by measuring the

expressions of three important antioxidant enzymes (Sod1, Sod2, and Catalase). HNE

protein adduct formation but not protein carbonylation rose significantly in the

diaphragm of the LPS group. Significant increases in both indices of protein oxidation

were observed in the MV and LPS+MV groups (Figure 11A-C). Both protein

carbonylation and HNE protein adduct formation values in the LPS+MV were

significantly higher than those of the LPS group (Figure 11A-C). Sod1 and Sod2

mRNA levels but not Catalase mRNA levels rose significantly in the LPS, MV and

LPS+MV groups compared to the control group (Figure 11D).

48

SECTION 4- DISCUSSION

The most important findings of this study observed in the diaphragm of rats:

1) Both LPS administration and prolonged MV attenuated muscle contractility.

Combining MV with LPS administration resulted in additional decline in muscle

contractile performance.

2) Muscle fiber atrophy was evident in response to LPS administration and prolonged

MV. Combining the two had no additional effect on the development of muscle

atrophy.

3) Proteasome, calpain, caspase-3 and the autophagy proteolytic pathways were

activated in the LPS and MV groups. Combining prolonged MV with sepsis resulted

in the potentiation of autophagy pathway but not proteasome, calpain and capase-3

activation.

4) The AKT and mTORC1 pathways (inhibitors of proteolytic pathways and

activators of protein synthesis) were activated in response to LPS administration but

not by prolonged MV. Combining sepsis with prolonged MV resulted in attenuation

of AKT and mTORC1 activation compared to sepsis alone.

5) The AMPK pathway (activator of autophagy) is inhibited in response to LPS

administration and prolonged MV. Combining sepsis with prolonged MV resulted in

a milder degree of AMPK inhibition compared to LPS administration alone.

6) Oxidative stress develops in response to LPS administration and prolonged MV.

Combining sepsis and prolonged MV resulted in potentiation of oxidative stress

development compared to LPS administration alone.

49

The major finding of the current study is that diaphragm force in septic

animals further deteriorated when prolonged MV was applied to these animals. This

is the first report of strong potentiating effect of prolonged MV on sepsis-induced

diaphragm contractile dysfunction. Indeed until now, previous studies which assessed

the effect of MV on diaphragm dysfunction in septic animals applied MV

concomitantly with the induction of sepsis. As a consequence, diaphragm function in

these experiments was relatively intact when prolonged MV was applied. In our

study, we used a more clinically relevant model in which humans with sepsis are

mechanically ventilated in response to the development of acute lung injury or

multiple organ failure. To simulate this clinical condition, we initiated the septic

process in our animals by injecting LPS 12h prior to the application of MV and hence

diaphragm force generation was already weakened in response to 12h of sepsis prior

to the application of prolonged MV. In previous studies, short-term MV (4h)

prevented sarcolemmal damage and significantly improved diaphragm force

production (40) while long-term MV further reduced diaphragm force after 12h (34)

or did not affect diaphragm function after 5 days of MV (93). The beneficial effect of

short-term MV on the diaphragm was attributed to the abrogation of the harmful

interaction between oxidative stress and biochemical stresses imposed on the

sarcolemma (40). Discrepant results regarding the long-term effects of MV and sepsis

on diaphragm contractile function can be explained in part to different doses of LPS

used in various studies. Relatively low doses of LPS were used in the study of Ochala

et al (20-30 µg/kg) (93) compared to that of Demoule et al (34). The LPS dose used

in the latter study is similar to the one used in the present study. This LPS dose is

frequently used to induce acute sepsis. Hence, our data are in agreement with those of

50

Demoule et al (34). We believe that our model, in which MV is applied after the

development of sepsis-induced diaphragm weakness, is more clinically relevant since

this corresponds more closely to the clinical settings of septic patients in the ICU.

The mechanisms involved in the worsening of diaphragm force generation in

the LPS+MV compared to the LPS or MV groups remain unclear. One possible

mechanism is hemodynamic alterations including hypotension associated with sepsis

and prolonged MV that may lead to poor diaphragm perfusion and depressed force

generation. However, we can exclude hypotension as a possible contributor to the

impaired diaphragmatic function in the LPS+MV group since arterial blood pressure

was similar in the LPS+MV compared to that of the LPS group (Table 2). Another

likely mechanism behind worsening of diaphragm force generation in the LPS+MV

group is metabolic acidosis and/or respiratory acidosis which develop during the

course of sepsis or prolonged MV in humans. In this study the mean pH in all groups

was above the threshold value of pH of 6.8 (Table 2), a value that is known to

severely depress diaphragm force generation (29). Another factor that may explain

worsening of diaphragm force generation in the LPS+MV group is the hypoxia which

is known to negatively influence muscle force generation. However, arterial PO2 is

significantly lower in the LPS group compared to the MV and MV+LPS groups

(Table 2). This observation renders hypoxia to be an unlikely factor to explain further

decline in diaphragm force generation in the LPS+MV group. We should emphasize

that supplemental O2 was delivered to the MV and LPS+MV groups since both

groups were mechanically ventilated while the LPS group animals were

spontaneously breathing and hence they did not receive supplemental O2. Finally,

anesthesia levels were the lowest in the LPS+MV group (Table 2) which would be

51

expected to positively influence diaphragm contractility. However diaphragm force

was the lowest in that group thereby ruling out any effect of anesthesia.

Prolonged MV and sepsis are known to independently activate the different

proteolytic systems in the diaphragm such as the calpain, caspase-3 and the

proteasome systems (35; 79; 122; 126). In the current study the calpain and caspase-3

system are unlikely to play a major role in the worsening of diaphragm contractile

dysfunction in the LPS+MV group compared to values measured in the LPS group

since activity levels of these two proteolytic pathways in the diaphragm of the

LPS+MV group were not different than the control group (Figure 7). We found that

the activity of the proteasome pathway was significantly elevated in the diaphragm of

the LPS, MV and LPS+MV groups as indicated by the rise in protein ubiquitin

conjugate formation (Figure 5) and the upregulation of UBC4 ubiquitin conjugase and

the expression of Atrogin-1 and Murf-1 E3 ligases (Figure 6). The negative

correlations observed between diaphragm force and Murf-1 or Atrogin-1 levels

emphasizes the importance of these changes and suggest that the impaired diaphragm

force in the LPS+MV group might be related to an activation of the proteasome

system. Evidence exists that degradation of diaphragm contractile proteins is achieved

by activation of this pathway (135). Activation of the proteasome system, in this

study, might be triggered by the elevated diaphragmatic IL-6 levels. IL-6 has indeed

been implicated as an important stimulus to initiate muscle atrophy and is known to

induce Atrogin-1 expression (9). This was also observed in a study of Van Hees et al

in which IL-6 was shown to play a prominent role in the induction of atrophy in

septic shock patients and in the activation of Atrogin-1 and MuRF-1 (135). It is well

known that sepsis and prolonged MV independently may trigger cytokine release into

52

the circulation. We observed an elevation of circulating and diaphragmatic IL-6 levels

in LPS+MV group compared to LPS or MV groups. This is in agreement with a

previous study in which LPS treated mice ventilated for 6h showed increased

circulating cytokines, including IL-6 (92). A possible explanation for the fact that IL-

6 is increased in the LPS+MV group only is that neutrophils and monocytes are

primed by LPS in the systemic circulation and will be further activated by mechanical

ventilation (stretch) in the pulmonary circulation, leading to a systemic inflammatory

response. Indeed stretch in combination with LPS has been shown to activate pro-

inflammatory pathways through separate but complementary mechanisms (5). We

also observed an increase in serum IL-1 in the LPS group only. Sepsis is known to

result into inflammation accompanied with increases of IL-1. On the other hand, IL-

1 was not increased in the diaphragm after prolonged MV (112). Remarkably, in the

LPS+MV group IL-1 levels were similar to the ones in the MV group, despite the

presence of sepsis. This suggests that in this group, prolonged MV or the treatments

during MV may play a role. In this regard, the fluid replacement therapy that was

used in the LPS-CMV animals might play a role.

We should emphasize that activation of the proteasome alone doesn’t fully

explain the worsening of diaphragm force generation in the LPS+MV compared to

the LPS alone since the degree of proteasome activation (protein ubiquitination,

UBC4 protein levels, Atrogin-1 and Murf-1 mRNA levels) is similar among the LPS

and LPS+MV groups (Figures 5 and 6). Furthermore, the degree of muscle atrophy as

measured by cross sectional areas was also similar in these two groups and hence

muscle atrophy doesn’t explain further deterioration of diaphragm contractile

53

dysfunction in the LPS+MV group compared to the LPS group (Figure 2). It is

possible that the fourth proteolytic system, namely, autophagy-lysosome pathway,

which regulate not only muscle mass but mitochondrial quality control and hence

muscle energetics may be responsible for worsening of diaphragm force dysfunction

in the LPS+MV group. Our results indicate that several markers of autophagy such as

LC3B protein lipidation (conversion of LC3B-I to LC3B-II) and expression levels of

several genes involved in autophagosome formation and recycling of the

mitochondria (Gabarapl1, Ambra1, Uvrag and PARKIN) are significantly greater in

the diaphragm of the LPS+MV compared to the LPS group (Figures 8 and 9). These

results are strong indicators of potentiation of autophagy in the diaphragm in response

to combination of prolonged MV with sepsis compared to sepsis alone. Interestingly,

the induction of autophagy in the LPS, MV and LPS+MV groups develops despite the

fact that two strong inhibitors of autophagy (the AKT and mTORC1 pathways) are

strongly activated particularly in the LPS group and that a strong stimulator of

autophagy (AMPK pathway) is actually inhibited in these groups (Figure 10). We

attribute the induction of autophagy in the LPS, MV and LPS+MV group in the

current study to other factors such as mitochondrial dysfunction and the development

of oxidative stress (see below).

The question of whether excessive autophagy plays an important role in the

deterioration of diaphragm force generation in the LPS+MV group or plays a

protective role in removing damaged mitochondria, protein aggregates and lipid

globules remains unanswered in the current study. In skeletal muscles, autophagy has

been shown to be a critical regulator of protein homeostasis and mitochondrial quality

(82; 108; 109). Recent studies have also revealed the following: a) autophagy is

54

significantly induced by catabolic stimuli such as starvation, denervation and sepsis

(87); b) the autophagic contribution to total muscle protein degradation can be as high

as the proteasomal contribution (80; 150); c) excessive autophagy fully accounts for

the atrophy that is triggered by oxidative stress (38); d) mitochondrial abnormalities

trigger significant increases in autophagy and atrophy (107). On the basis of these

findings, we propose that excessive autophagy that was observed in the diaphragm of

septic animals exposed to prolonged MV may participate in depressing diaphragm

contractile function through excessive recycling of mitochondria and thereby

reducing mitochondrial density and impairs muscle energy supplies. Excessive

autophagy can also interfere with proper muscle contractile force generation through

degradation of proteins that are critically involved in the signaling processes of

muscle contraction and those involved in homeostasis of myofilament protein

interactions. So far, little is known about exact protein targets of autophagic

proteolytic pathway in skeletal muscles. Our research group have accumulated

unpublished results indicate that selective inhibition of autophagy in septic mice

restores the decline in diaphragm and limb muscle contractile function indicating that

excessive autophagy in septic mice may contribute to the decline in skeletal muscle

performance. Similar experiments are needed to test the contribution of excessive

autophagy to worsening of diaphragm contractile function in animals which are septic

and exposed to prolonged MV.

The significant rise in the expression of p62 and PARKIN which are involved

in the targeting of depolarized and dysfunctional mitochondria by the

autophagosomes (mitophagy) in the diaphragm of the LPS+MV group is an indicator

that mitochondrial dysfunction deteriorated when septic animals are exposed to

55

prolonged MV. Mitochondrial dysfunction is a known complication in sepsis. Several

reports have confirmed that complex I and IV activities of oxidative phosphorylation

complexes in the ventilatory and limb muscles are significantly decreased in humans

and animals with sepsis (17; 20; 21; 24; 30; 31; 45; 48; 136). However, the

underlying causes of mitochondrial enzyme dysfunction in sepsis remain under

debate. Some investigators have implicated oxidative modification of mitochondrial

enzymes; others have claimed that reduction in mitochondrial content is more

important (44; 56). It should be emphasized that reduction in mitochondrial content in

septic muscles leads to decreased high-energy phosphates and increased lactates. This

occurs at the same rate in the ventilatory and limb muscles of septic patients (43).

Since mitochondrial content is regulated by a balance between biogenesis and

recycling, it might be concluded that decreased mitochondrial content in septic

muscle is due to decreased synthesis of mitochondrial proteins. However, Fredriksson

et al. (43) have found that in-vivo mitochondrial protein synthesis does not

significantly decrease in muscles of septic patients. They therefore suggest that

decreased mitochondrial content is a result of enhanced degradation and recycling.

This proposal is designed to provide evidence that will end the debate.

Another important mechanism that may explain the substantial induction of

autophagy and worsening of diaphragm contractile dysfunction in the MV+LPS

group is oxidative stress. Our results indicate that two indirect markers of oxidative

stress (protein carbonylation and HNE protein adduct formation) are potentiated when

MV is combined with sepsis (Figure 11) suggesting that oxidative stress is worsened

in the LPS+MV compared to the LPS group alone. Reactive oxygen species (ROS),

including O2- , H2O2, and HO

-, are produced at relatively low rates in resting muscle

56

fibres (37; 53; 69; 102; 105). Increased ROS levels have been extensively

documented in the ventilatory and limb muscles of humans and animals with sepsis

(7; 42; 96; 117; 121; 125). Increases in ROS production and/or decreases in

antioxidant levels result in accumulation of ROS and the development of oxidative

stress. Oxidative stress alters skeletal muscles in sepsis by inducing mitochondrial

dysfunction (17) and by modifying critical proteins such as creatine kinase and

myosin, increasing their degradation and inhibiting their activity (11; 12; 58).

Moreover, there is strong evidence that oxidative stress in septic muscles enhances

protein degradation by stimulating calpains, caspases, and proteasome (84; 123; 124;

126). Several authors have shown that pre-treatment of septic animals with

antioxidant enzymes and free radical scavengers reduces oxidative stress and

improves muscle function (117; 120; 122; 129).

Several reports have confirmed that autophagy is activated by oxidative stress.

Selective attenuation of mitochondrial ROS strongly attenuates stress-induced

autophagy, confirming the importance of mitochondria to the process (27; 147). ROS

influence autophagy through several mechanisms of action including: activation of

AMPK and inhibition of mTORC1 (113). This mechanism is not likely to be

important in our study since AMPK is actually inhibited and mTORC1 is activated in

the diaphragm of the LPS+MV group (Figure 10). Another mechanism through which

ROS enhance autophagy is the recruitment of BNIP3 to the mitochondria as a BCL2-

binding competitor of BECLIN1, a trigger of autophagy (113) and through activation

of FoxO transcription factors, which regulate many autophagy-related genes,

including LC3, GABARAP, and BNIP3 (150). Finally, oxidative stress may activate

57

mitochondrial permeability transition pore, which results in loss of membrane

potential and recruitment of PARKIN and autophagosomes to the mitochondria (91).

In summary, we show here that diaphragm dysfunction is worsened when

septic animals are exposed to prolonged mechanical ventilation and that this

worsening of contractile dysfunction is associated with excessive oxidative stress and

substantial induction of autophagy and mitochondrial recycling pathways. We

propose that future experiments should be aimed at directly investigating the role of

autophagy in worsening of diaphragm dysfunction when sepsis is combined with

prolonged MV and additional studies are also required to document the contribution

of oxidative stress to the regulation of diaphragm contractile function, proteolysis and

autophagy in septic animals exposed to prolonged MV.

58

SECTION 5- TABLES

Table 1: Primers used for real-time PCR experiments to detect the expression of

various mRNAs in the diaphragm of the four groups of animals.

Gene

Atrogin-1 Forward 5’-TGCTCAGTGAAGACCGGCTA -3’

Reverse 5’-TTGGGTAACATCGCACAAGC -3’

Murf-1 Forward 5’-GGGAACGACCGAGTTCAGAC -3’

Reverse 5’-GCGTCAAACTTGTGGCTCAG -3’

Nedd4 Forward 5’-GACCAAGCCCTGAGGATGAC -3’ Reverse 5’-TTCTCAGGGGACTCGTGGTT -3’

Gabarapl1 Forward 5’- TAAAGAGGACCACCCCTTCG-3’ Reverse 5’- CGGAGGGCACAAGGTACTTC-3’

Ambra1 Forward 5’-GGAGGGGTTTTCCATCATCA -3’

Reverse 5’-AGGCTCTGATCCAGCTCCTG -3’

Uvrag Forward 5’-GGCCTTCCTGCATAAGCAAC -3’

Reverse 5’-CTCCTTCCTCAGCTCCCTCA-3’

Ulk1 Forward 5’CACTGCGTGGCTCACCTAAG-3’

Reverse 5’- AGCCAACAGGGTCAGCAAAT-3’

Ulk2 Forward 5’- TTGCAATGGTGGAGATCTGG-3’

Reverse 5’- ATCCCTTTGCTGTGCAGGAT-3’

Sod1 Forward 5’-GCGTCATTCACTTCGAGCAG -3’

Reverse 5’-CCTGCAGTGGTACAGCCTTG -3’

Sod2 Forward 5’-GTGGGAGTCCAAGGTTCAGG -3’

Reverse 5’-AGTAAGCGTGCTCCCACACA -3’

Catalase Forward 5’-CGGGTTGCCTAGAAGGACAG -3’

Reverse 5’-ACAGCCCTGATTGCCTTGAT -3’

-Actin Forward 5’-TGTGGCATCCATGAAACTACATT -3’ Reverse 5’-AGGAGCAATGATCTTGATCTTCA -3’

59

Table 2: Anesthesia levels, arterial blood pressure and blood gas data measured after

24h of experimentation period in the LPS, MV and LPS+MV groups. Values are

means ± SD. $ p<0.05 compared with the LPS or MV groups, @ p<0.05 compared to

the MV group. NA: not applicable

MV group LPS group LPS+MV group

Anesthesia (mg/h/100g) 1.63± 0.20 NA 0.71± 0.23 @

Mean Arterial Pressure

(mmHg) 141 ± 28 $ 92 ± 23 74 ± 15

pH 7.451 ± 0.16 7.456 ± 0.04 7.246 ± 0.11$

paO2 (mm Hg) 131 ± 37 76 ± 37 @

117 ± 40

paCO2 (mm Hg) 28 ± 11 23 ± 8 27 ± 12

60

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SECTION 7- FIGURE LEGENDS

Figure 1: Diaphragm maximum twitch force, maximum tetanic force and the force-

frequency relationships in the control (spontaneously breathing), LPS, MV and

LPS+MV groups. Values are expressed as means ± SE. *p<0.05 compared to the

control values. #p<0.05 compared to the LPS group.

Figure 2: Changes in diaphragm CSA of type I, IIa and IIx/b fibers measured in the

control, LPS, MV and LPS+MV groups. Values are meansSEM. *P<0.05

compared to the control values (spontaneously breathing rats).

Figure 3: Plasma IL-1 (A) and plasma IL-6 (B) values measured after 24h of

experimental period in the control, LPS, MV and LPS+MV groups. Values are means

SEM. *p<0.05 compared to the control values. #p<0.05 compared to the LPS

group.

Figure 4: Diaphragm TNF, IL-1 and IL-6 levels measured in the control, LPS,

MV and LPS+MV groups. Values are means SEM. *p<0.05 compared to the

control values. #p<0.05 compared to the LPS group.

Figure 5: A) Representative immunoblot of protein ubiquitination in the diaphragm

of the four groups of animals.

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B: Optical densities of protein ubiquitin conjugate formation in the diaphragm

of the four groups of animals. Values (means SEM) are expressed as fold change

from control values. *p<0.05 compared to the control values.

Figure 6: A) Representative immunoblots of ubiquitin conjugases UBC2 and UBC4

and the subunits of the 20S proteasome in the diaphragm of the four groups of

animals.

B: Optical densities of UBC2, UB4 and subunits of the 20S proteasome in

the diaphragm of the LPS, MV and LPS+MV groups of animals. Values (means

SEM) are expressed as fold change from the control values.

C: mRNA levels of muscle-specific E3 ligases Atrogin-1, Murf-1 and Nedd4

in the diaphragm of the LPS, MV and LPS+MV groups. Values (means SEM) are

expressed as fold change from control values. *p<0.05 compared to the control

values.

Figure 7: Calpain and caspase-3 activities (derived from immunoblotting of -II

SPECTRIN protein) in the four groups of animals. Values ((means SEM) are

expressed as the ratio of cleaved -II SPECTRIN over total SPECTRIN optical

densities. *p<0.05 compared to the control values.

Figure 8: A) Representative immunoblots of LC3B and -ACTIN proteins in the

diaphragm of the LPS, MV and LPS+MV groups. LC3B-I refers to cytosolic form

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of LC3B protein, LC3B-II refers to lipidated form of LC3B protein. LC3B-II is

incorporated into autophagosome membranes.

B) Optical densities of LC3B-II/LC3B-I ratios and LC3B-II detected in the

diaphragm of the LPS, MV and LPS+MV groups. Values (means SEM) are

expressed as fold changes from the control values. *P<0.05, compared to control

values. # *P<0.05, compared to the LPS group.

Figure 9: A-B) Representative immunoblots of SQSTM1 (p62), BECN1, BNIP3,

ATG7, PARKIN, and -TUBULIN in the diaphragm of the four groups of animals.

C) Protein expressions of SQSTM1, BECN1, PI3KCIII, BNIP3, and PARKIN in

the diaphragm of the LPS, MV and LPS+MV groups. Values (means SEM) are

expressed as fold change from control values. *P<0.05, compared to the control

group. #P<0.05 compared to the LPS group.

D) mRNA expressions of Gabarapl1, Ambra1, Uvrag, Ulk1 and Ulk2 in the

diaphragm of the LPS, MV and LPS+MV groups. Values (means SEM) are

expressed as fold change relative to the control group. *P<0.05, compared to the

control group. #P<0.05 compared to the LPS group.

Figure 10: A-C-E) Representative immunoblots of phosphorylated and total AKT,

P70S6K1, and AMPK in the diaphragm of the LPS, MV and LPS+MV groups.

B-D-E) Protein expressions of phosphorylated and total AKT, P70S6K1, and

AMPK (normalized to control values) in the diaphragm of the LPS, MV and

90

LPS+MV groups. *P<0.05, compared to the control group. #P<0.05 compared to

the LPS group.

Figure 11: A-B) Representative immunoblots of protein carbonyl formation and

HNE protein adduct formation in the diaphragm of the four groups of rats.

B) Total optical densities of protein carbonyls and HNE protein adduct formation

(normalized to the control group) in the diaphragm of the LPS, MV and LPS+MV

groups.

C) mRNA expressions of Sod11, Sod2, and Catalase in the diaphragm of the LPS,

MV and LPS+MV groups. Values (means SEM) are expressed as fold change

from the control values. *P<0.05, compared to control subjects. #P<0.05 compared

to the LPS group.

91

SECTION 8- FIGURES

FIGURE 1

92

FIGURE 2

93

FIGURE 3

94

FIGURE 4

95

FIGURE 5

96

FIGURE 6

97

FIGURE 7

98

FIGURE 8

99

FIGURE 9

100

FIGURE 10

101

FIGURE 11