effect of prolonged mechanical ventilation on sepsis...
TRANSCRIPT
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
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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
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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
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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).
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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
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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.
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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.