ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2017

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1368

Muscle Wasting in a Rat ICUModel

Underlying Mechanisms and Specific InterventionStrategies

HEBA SALAH

ISSN 1651-6206ISBN 978-91-513-0061-0urn:nbn:se:uu:diva-328596

Dissertation presented at Uppsala University to be publicly examined in Seminarierum kliniskneurofysiologi, Ing 85, Akademiska sjukhuset, Uppsala, Monday, 23 October 2017 at 09:00for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will beconducted in English. Faculty examiner: Professor Sven Tågerud (Linnaeus University).

AbstractSalah, H. 2017. Muscle Wasting in a Rat ICU Model. Underlying Mechanisms and SpecificIntervention Strategies. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1368. 61 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-513-0061-0.

Critical care has undergone several developments in the recent years leading to improvedsurvival. However, acquired muscle weakness in the intensive care unit (ICU) is an importantcomplication that affects severely ill patients and can prolong their ICU stay. Critical illnessmyopathy (CIM) is the progressive decline in the function and mass of the limb muscles inresponse to exposure to the ICU condition, while ventilator-induced diaphragm dysfunction(VIDD) is the time dependent decrease in the diaphragm function after the initiation ofmechanical ventilation. Since the complete underlying mechanisms for CIM and VIDD are notcompletely understood, there is a compelling need for research on the mechanisms of CIM andVIDD to develop intervention strategies targeting these mechanisms. The aim of this thesis wasto investigate the effects of several intervention strategies and rehabilitation programs on musclewasting associated with ICU condition. Moreover, muscle specific differences in response toexposure to the ICU condition and different interventions was investigated. Hence, a rodentICU model was used to address the mechanistic and therapeutic aspects of CIM and VIDD. Theeffects of heat shock protein 72 co-inducer (HSP72), BGP-15, on diaphragm and soleus for ratsexposed to different durations of ICU condition was investigated. We showed that 5 and 10days treatment with BGP-15 improved diaphragm fiber and myosin function, protected myosinfrom posttranslational modification, induced HSP72 and improved mitochondrial function.Moreover, BGP-15 treatment for 5 days improved soleus muscle fibers function, improvedmitochondrial structure and reduced the levels of some ubiquitin ligases. In addition to BGP-15treatment, passive mechanical loading of the limb muscles was investigated during exposure tothe ICU condition. We showed that mitochondrial dynamics and mitophagy gene expressionwas affected by Mechanical silencing while mechanical loading counteracted these effects.Our investigation for other pathways that can be involved in muscle wasting associated withICU condition showed that the Janus kinase 2/ Signal transducer and activator of transcription3 (JAK2/STAT3) pathway is differentially activated in plantaris, intercostals and diaphragm.However, further studies are required with JAK2/STAT3 inhibitors to fully examine the role ofthis pathway in the pathogenesis of CIM and VIDD prior to translation to clinical research.

Keywords: BGP-15, critical illness myopathy, janus kinase, intensive care unit, mechanicalloading, muscle wasting, signal Transducer and Activator of Transcription, ventilator-induceddiaphragm dysfunction

Heba Salah, Department of Neuroscience, Clinical Neurophysiology, Akademiska sjukhuset,Uppsala University, SE-75185 Uppsala, Sweden.

© Heba Salah 2017

ISSN 1651-6206ISBN 978-91-513-0061-0urn:nbn:se:uu:diva-328596 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-328596)

To my parents To Derar

To my Summer

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Salah H., Li M., Cacciani N., Gastaldello S., Ogilvie H., Akkad H.,

Namuduri AV., Morbidoni V., Artemenko KA., Balogh G., Martinez-Redondo V., Jannig P., Hedström Y., Dworkin B., Bergquist J., Ruas J., Vigh L., Salviati L., Larsson L. (2016) The chaperone co-inducer BGP-15 alleviates ventilation-induced diaphragm dysfunction. Sci-ence translational medicine, 8(350):350ra103.

II Cacciani N.*, Salah H.*, Li M1, Akkad H., Ogilvie H., Backeus A.,

Hedstrom Y., Larsson L. Does chaperone co-inducer BGP-15 mitigate the contractile dysfunction of the soleus muscle in a rat ICU model? Manuscript.

III Corpeno Kalamgi R., Salah H., Gastaldello S., Martinez-Redondo V.,

Ruas JL., Fury W., Bai Y., Gromada J., Sartori R., Guttridge DC., Sandri M., Larsson L. Mechano-signalling pathways in an experi-mental intensive critical illness myopathy model. (2016) The Journal of physiology, 594(15):4371-88.

IV Salah H., Fury W., Gromada J., Tchkonia T., Kirkland JL., Larsson L.

Muscle specific differences in activation of the Janus kinase 2/ Signal Transducer and Activator of Transcription 3 following long-term me-chanical ventilation and immobilization in rats. Submitted.

* Contributed equally to this study.

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11 Skeletal Muscle ........................................................................................ 11 

Actin .................................................................................................... 11 Myosin ................................................................................................. 12 Muscle Contraction .............................................................................. 12 

Mitochondria ............................................................................................ 15 Mitochondria structure ......................................................................... 15 Respiratory chain complexes ............................................................... 15 Mitochondria are dynamic organs ....................................................... 16 Mitochondria biogenesis ...................................................................... 16 Mitochondria in skeletal muscle .......................................................... 17 

JAK/STAT Pathway ................................................................................. 18 Janus Kinase Family ............................................................................ 18 Signal Transducer and Activator of Transcription (STATs) ............... 18 Regulation of JAK/STAT pathway ..................................................... 19 STAT3 is linked to muscle atrophy ..................................................... 19 

Cellular mechanisms of muscle atrophy .................................................. 19 The ubiquitin proteasome system (UPP) ............................................. 20 Autophagy lysosomal pathway ............................................................ 20 Inflammatory cytokines ....................................................................... 21 Apoptosis ............................................................................................. 22 

Muscle wasting in intensive care unit ...................................................... 23 Critical Illness Myopathy (CIM) ......................................................... 23 Ventilator-Induced Diaphragm Dysfunction ....................................... 24 Therapeutic approaches ....................................................................... 25 

Aims of the thesis.......................................................................................... 27 

Material and Methods ................................................................................... 28 Animals (Papers I, II, III and IV) ............................................................. 28 Muscle biopsy (Papers I, II, III and IV) ................................................... 28 Membrane permeabilization and single muscle fiber contractile measurements (papers I and II) ................................................................ 29 Modified single fiber myosin in vitro motility assay for speed and force measurements (papers I and II) ....................................................... 29 Transmission electron microscopy (papers I and II) ................................ 29 

Respiratory chain enzymatic activity (paper I) ........................................ 30 Lipidomics, proteomics, and mass spectroscopy (Paper I) ...................... 31 Subcellular fractioning (paper III) ............................................................ 32 Western blotting ....................................................................................... 32 Gene expression analysis ......................................................................... 33 Statistics (paper I, II, III and IV) .............................................................. 35 

Results and Discussion ................................................................................. 36 

Conclusions ................................................................................................... 47 

Acknowledgments......................................................................................... 49 

References ..................................................................................................... 51 

Abbreviations

ATP Adenosine triphosphate MyHC Myosin heavy chain Ach Acetylcholine ADP Adenosine diphosphate OxPhos Oxidative phosphorylation ROS Reactive oxygen species DRP1 Dynamin-related protein 1 FIS1 Mitochondrial fission protein 1 OPA1 Optic atrophy 1 MFN1 Mitofusin 1 MFN2 Mitofusin 2 PPARγ Peroxisome proliferator-activated receptor gamma PGC1 PPARγ coactivator 1 COXIV Cytochrome C oxidase IMF Intermyofibrillar mitochondria SS Subsarcolemmal mitochondria JAK Janus kinase STAT Signal transducer and activator of transcription TYK Tyrosine kinase CIS Cytokine-inducible SH2 proteins JAB JAK2 binding protein SOCS Suppressor of cytokine signaling SSI STAT induced STAT inhibitors PIAS Protein inhibitor of activated STAT IL-6 Interleukin-6 UPP ubiquitin proteasome pathway E1 Ubiquitin activating enzyme E2 Ubiquitin-conjugating enzyme E3 Ubiquitin ligase MuRF1 Muscle RING-finger protein-1 MyBP-C Myosin binding protein C PTEN Phosphate and tensin homolog PINK1 PTEN-induced putative kinase 1 Bnip3 BCL2/adenovirus E1B 19kDa protein-interacting protein 3 NF-kB nuclear factor kappa-light-chain-enhancer of activated B

cells

TNF-α Tumor necrosis factor alpha IKKB Inhibitor of nuclear factor kappa B kinase subunit beta IFN-γ Interferon gamma C/EBPδ CCAAT-enhancer-binding protein delta Apaf-1 Apoptotic protease activating factor 1 AIF Apoptosis inducing factor EndoG Endonuclease G mPTP Mitochondrial permeability transition pore ICUAW Intensive care unit acquired weakness CIM Critical illness myopathy CIP Critical illness polyneuropathy CSA Cross sectional area VIDD Ventilator-induced diaphragm dysfunction CMV Controlled mechanical ventilation HSP Heat shock protein

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Introduction

Skeletal Muscle Skeletal muscle is a dynamic and plastic tissue comprising 30-50% of human body mass. Besides being important to generate motion and force, skeletal muscles regulate body temperature, and secrete several cytokines and growth factors. In humans, skeletal muscle contains 50-70% of body proteins making skeletal muscle a storage site for amino acids. The general structure of skeletal muscle consists of connective tissue and bundles of individual skeletal muscle cells that are called myofibers. The myofiber is usually composed of myofi-brils that contain several myofilaments. When arranged in a specific manner, the myofilaments form the sarcomere which is the basic contractile unit in the muscles. In addition to myosin and actin, the sarcomere and the sarcoplasm contain several proteins that are important for energy release, and the regula-tion of muscle contraction like tropomyosin and troponin. All skeletal muscles in the body are continuously remodeled to match their demand, thus, the di-ameter, length, force, and vascular supply can be changed. In general, muscle mass is regulated by the balance between protein synthesis and degradation and both processes are labile to nutritional status, hormonal balance, levels of physical activity, and the presence of certain diseases or injuries.

Actin Actin is a highly-conserved protein that constitutes around 20% of total cellu-lar proteins in muscle cells. Due to its ability to transform into 2 different isoforms, monomeric (G-actin) and filamentous (F-actin), actin plays an im-portant role in several cellular processes like motility, maintenance of cell shape, and regulation of transcription [1]. The three dimensional structure of an actin monomer is divided into 4 subdomains that are arranged in a way forming 2 clefts [2]. One of these clefts is for binding adenosine triphosphate (ATP) and Mg, while the other is made mostly of hydrophobic residues for binding actin binding proteins [3]. In skeletal muscle, G-actin monomers pol-ymerize to form the actin filaments that are associated with other proteins, tropomyosin and troponin, to regulate muscle contraction. In the relaxed state, tropomyosin blocks the active site on actin preventing it from binding to my-osin.

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Myosin Myosins are family of motor proteins involved in the transport of specific bi-omarkers, vesicles, and organelles in eukaryotic cells [4, 5]. The myosin su-perfamily is divided into at least twenty-four classes based on the head domain sequence similarities and domains organization [5-7]. They are made of 1000-2000 residues that are localized into different organs or cell types based on their structural and functional properties. The most common non-muscular myosin are cytoplasmic [5]. The myosin class that was first identified in mus-cle cells is myosin II. This conventional form of muscle myosin is made of two heavy chains, and 4 light chains [8]. The heavy chain of the myosin con-sists of 2 parallel α-helical rods with a globular domain (head) on the end of each rod [9]. The globular domains carry the ATPases i.e. the enzymatic active site for myosin that are activated in the sarcomere by binding to the actin fil-ament. Therefore, these domains contain binding sites for both actin and ATP [10]. The rest of the myosin head has a narrow nick that links it to the rod, and each neck carries regulatory and essential light myosin chains while the rod region forms the thick filament backbone in the sarcomere.

The myosin heavy chain (MyHC), where the catalytic and motor function of myosin are, is a highly conserved molecule [11]. Up to date, 13 genes have been identified for MyHC, of which at least 8 genes are expressed in skeletal muscles. The most important genes in skeletal muscles are: Myh7 which en-codes the slow fibers Myosin heavy chain I (MyHCI), Myh1, Myh2, and Myh4 which encodes the fast fibers MyHCIIx, MyHCIIa, and MyHCIIb, respec-tively [8].

Muscle Contraction Skeletal muscles are activated by the action potential that travels from the mo-tor nerve to the neuromuscular junction. At the junction, the motor neuron releases the neurotransmitter acetylcholine (ACh), in the synaptic cleft, which bind to special muscarinic receptors on the muscle fiber membrane. The bind-ing of ACh to its receptors opens ACh -gated channels that allow the entrance of Na+ in exchange for K+ initiating an action potential in the muscle fiber membrane that travels along the fiber. Later, the action potential is transferred into the sarcomeres through the T-tubules, invaginations of the sarcolemma, leading to the release of Ca2+ from the sarcoplasmic reticulum which initiates the attraction between actin and myosin [12, 13].

During relaxed conditions, tropomyosin blocks the myosin binding sites on actin. When Ca2+ is released from the sarcoplasmic reticulum, it binds tro-ponin C which in response undergoes a conformational change that moves with it the tropomyosin, and thus, uncover the myosin binding sites on actin. Before contraction, the myosin heads bind ATP that is later, hydrolyzed by the myosin ATPase activity into ADP and Pi. Later, the myosin heads bind to

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the myosin binding sites of actin when they become uncovered by Ca2+ re-lease. This binding between actin and myosin initiates a conformational change in the myosin heads tilting them towards the arm of the cross-bridge, and thus provides the power stroke for pulling the actin filament. In addition, this conformational change allows the release of the ADP and Pi from the myosin heads. Therefore, new ATP molecules can bind again and the heads detach from the actin (Figure 1) [14, 15].

In the presence of ADP, the myosin heads form a strong complex with actin [16], but when ATP binds to myosin, a weaker complex is formed with disor-dered catalytic and light chain binding domains of myosin. A structural change in these domains is caused by phosphate (Pi) release, which also generates force and starts a new cycle for the system [17, 18]. Force declines when the system spends long time in the weak binding state or when the strong binding state is weakened, while velocity declines if the system spends long time in strong binding states [16].

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Figure 1. Muscle contraction

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Mitochondria Mitochondria are oval-shaped organelles that have two membrane systems: an outer membrane and a highly folded inner membrane that forms cristae com-partments. A major function of mitochondria is the aerobic production of ATP in cells through the oxidative phosphorylation (OxPhos) machinery [19]. However, mitochondria are also involved in several biochemical tasks in cells like Signaling (through mitochondrial reactive oxygen species (ROS)) [20], apoptosis [21], and calcium signaling and storage [22]. Furthermore, mito-chondria contain several antioxidant enzymes that scavenger several types of free radicals that are produced in the cell.

In addition to their biochemical activities, mitochondria have dynamic ac-tivities, such as fusion and fission, biogenesis, and autophagy.

Mitochondria structure The mitochondria are separated from the cytoplasm by two membranes; the inner and the outer membranes. In normal mitochondria, the inner membrane lies closely opposed to the outer membrane and separated by a small inter-membrane space. At various locations, the inner membrane projects into the matrix forming cristae [23, 24], and thus the inner and outer membranes divide the mitochondria into 3 compartments. The innermost compartment is sur-rounded by the inner membrane and is called mitochondrial matrix. The ma-trix has a high pH (7.9-8)[25] to create a trans-membrane electrochemical gra-dient that derives the synthesis of ATP [26]. In addition, the matrix is the site for mitochondrial DNA replication, protein biosynthesis and several enzy-matic reactions. The second mitochondrial compartment is the intermitochon-drial space, which is a gap between the inner and the outer mitochondrial membranes. All the proteins that are imported from the cytoplasm into the matrix must pass through this space, and thus the protein translocases of the outer and inner membrane form supercomplexes that span the intermembrane space [26, 27]. The third compartment in mitochondria is the crista lumen. The cristae contain most of the fully assembled electron transport chain com-plexes and ATP synthases and it contains a large amount of cytochrome C [26]. Structural changes can be observed in mitochondria during apoptosis where internal membrane remodeling occur leading to the formation of vesic-ular compartments or swelling of the affected organelle [28].

Respiratory chain complexes Most enzymes of the respiratory chain are large multi-subunit protein assem-blies that contain several redox cofactors [29]. The respiratory chain consists of four complexes, Complexes I, II, III, and IV. Together with complex V, which is also called ATP synthase, they form the OxPhos system. In OxPhos,

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the electrons are transferred form NADH or FADH2 to O2 through these pro-tein complexes that are located in the mitochondrial inner membrane. During this transfer, protons are pumped out of the mitochondrial matrix, resulting in generation of a pH gradient and a transmembrane electrical potential that cre-ates a proton-motive force. When the protons move back through the enzyme complex V, ATP is synthesized [30].

The respiratory chain complexes in normal mitochondria can assemble to form supercomplexes, which are also called respirasomes [31]. In mammals, the supercomplexes are made from complexes I, III, and IV in different com-binations [32]. Supercomplexes are functionally important since they stabilize the individual complexes, enhance the electron transfer across the electron transport chain, and minimize the level of ROS production [33-35]. Oxidative stress affects supercomplex formation by modifying mitochondrial mem-branes environment as a consequence of lipid peroxidation, thus disrupting supercomplex assembly and reducing energy production [23, 36].

Mitochondria are dynamic organs In vivo, mitochondria form a complex of interconnected network. The archi-tecture of this mitochondrial network is dynamic because mitochondria fre-quently divide in a process called mitochondrial fission, and fuse in a process called mitochondrial fusion [37]. The equilibrium between mitochondrial fis-sion and fusion affects mitochondrial shape, size, and the degree of connec-tivity of the mitochondrial network. Mitochondrial fission is controlled by several mitochondrial proteins that include Dynamin related protein-1 (DRP1) and Fission 1 (FIS1), whereas proteins regulating mitochondrial fusion in-clude optic atrophy 1 (OPA1) and mitofusin 1 and 2 (MFN1/2) [37, 38]. MFN1 and MFN2 are required for the fusion of both outer and inner mito-chondrial membrane, while OPA1 is required for the fusion of the inner mito-chondrial membrane and thus, important for the regulation of cristae mem-branes [39]. However, a recent study showed that OPA1 was unable to tubu-late and fuse mitochondria lacking the outer membrane mitofusin 1 but not mitofusin 2 [40].

Mitochondria biogenesis Mitochondrial biogenesis is a very complex process which involves the coor-dination of two distinct sets of cellular DNAs. The first set is the nuclear DNA which encodes the majority of mitochondrial proteomes. The second set is the mitochondrial DNA that encodes 13 essential proteins for the oxidative phos-phorylation machinery [41, 42]. The biogenesis of mitochondria is a long term adaptive response [41]. It does not occur due to transient increase in cellular energy demands where there is only an increase in mitochondrial activity.

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Recently, a large number of transcription regulators for mitochondrial bio-genesis have been evaluated. PPARδ coactivator 1 family (PGC 1), mainly PGC-1α, plays an important role in the control of mitochondrial biogenesis and function by integrating physiological signals and enhancing the function of diverse transcription factors acting at mitochondrial genes [43, 44]. In ad-dition, PGC-1α binds a large complement of transcription factors that are di-rectly associated with mitochondria respiratory function or that act directly on the expression of the respiratory chain [45]. For instance, ectopic PGC-1α ex-pression in cultured cells increased cytochrome c oxidase (COXIV) and cyto-chrome c protein levels, as well as steady state levels of mitochondrial DNA [46]. The role of PGC in mitochondrial biogenesis in mice has been widely studied in various tissue types. For instance, transgenic expression of PGC-1α in skeletal muscle leads to an increase in mitochondrial content, increased ex-pression of mitochondrial genes, and enhanced exercise performance [47].

Mitochondria in skeletal muscle Skeletal muscle cells are densely populated with mitochondria due to their high metabolic demand and the requirement for rapid and sustained ATP pro-duction. The skeletal muscle tissue has two subpopulations of mitochondria: intermyofibrillar mitochondria (IMF) that are positioned among myofibrils and subsarcolemmal mitochondria (SS). IMF usually accounts for almost 80% of skeletal muscles mitochondria [48]. IMF and SS differ in their protein con-tent and function. For instance, IMF express higher levels of OxPhos protein complexes, and have superior mitochondrial coupling, and thus, IMF are highly specialized in calcium handling and energy production for contractile activity [49, 50]. SS mitochondria provide energy for membrane related events [51].

As the mitochondria are the main producers of cellular ATP and free radi-cals, defects in mitochondria function are thought to cause a decline in skeletal muscle function. Muscle inactivity is also associated with significant changes in mitochondrial structure and biochemical activities. In 1972, a study re-ported a time dependent impairment in mitochondrial function that began within 24 h following the beginning of muscle inactivity (36). Thereafter, many studies have confirmed that prolonged skeletal muscle inactivity results in changes in mitochondrial morphology, decreased expression of mitochon-drial proteins, mitochondrial respiratory dysfunction, and increased mitochon-drial ROS production.

Emerging data supported the fact that increased ROS production is a major contributor to the mitochondrial damage and dysfunction that is associated with prolonged skeletal muscle inactivity. Increased ROS production in-creases proteolysis by activating calpain and caspase 3, and increasing the ex-pression of E3 ligases [52, 53]. In addition, ROS increases muscle protein ox-idation leading to their rapid degradation by cellular proteases [54]. ROS also

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causes permeabilization of mitochondrial outer membrane leading to the re-lease of proapoptotic factors such as AIF and cytochrome c [55].

JAK/STAT Pathway The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a signaling pathway for a wide range of cytokines and growth fac-tors. The JAK/STAT pathway stimulates cell proliferation, differentiation, mi-gration and apoptosis, and plays an important role in other cellular processes such as hematopoiesis, immune development, and adipogenesis [56].The sig-nal in the JAK/STAT pathway is initiated by the binding of an extracellular ligand into a membrane receptor. The ligand binding results in multimeriza-tion of the receptor which places their associated JAKs proteins into proxim-ity, and later causes the phosphorylation and thus, activation of JAKs. The phosphorylated JAKs act as a docking site for STATs, which are usually lo-cated in the cytoplasm. Later STATs are phosphorylated by JAKs and the phosphorylated STATs polymerize and move to the nucleus where they regu-late the transcription of several downstream genes [57].

Janus Kinase Family Janus Kinase (JAK) is a family of tyrosine kinases that transduces cytokines mediated signals through the JAK/STAT pathway. There are 4 members in the JAK family; JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) [58]. JAKs range from 120-140 kDa in size and have seven defined homology do-mains, one of them is important for the enzymatic activity of the JAK and contains a dual tyrosine residues. The phosphorylation of these residues acti-vates JAK protein by initiating conformational changes of the JAK protein to facilitate its interaction with several substrates [59].

Signal Transducer and Activator of Transcription (STATs) STAT family of proteins regulates several processes inside the cells like growth, survival, and differentiation. Seven members of the STAT family have been identified; STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 [60]. STAT proteins were originally described as latent cytoplas-mic transcription factors that require phosphorylation for nuclear retention. The unphosphorylated STATs usually shuttles between cytosol and the nu-cleus waiting for the activating signal. Once activated, STAT molecules di-merize and bind to DNA recognition motif called gamma activated sites (GAS) in the promotor region of cytokine-inducible genes and regulate the transcription of these genes [59]. Despite the diversity of receptors that act

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through the JAK/STAT pathway, there is no simple direct relationship be-tween JAK and STAT family members. However, in general, STAT3 and STAT5A/B promote oncogenesis, whereas STAT1 activation has opposing effects [61].

Regulation of JAK/STAT pathway JAK/STAT pathway is regulated by both negative and positive feedbacks. Several negative regulators have been described for this pathway like Cyto-kine inducible SH2 proteins (CIS) [62], Jak2 binding proteins (JAB) [63], sup-pressor of cytokine signaling (SOCS) [64]or STAT induced STAT inhibitors (SSI) [65]. All these regulators are up-regulated upon stimulation with various ligands, and inhibit further signaling through the JAK/STAT pathway. An-other family of STAT inhibitors are the protein inhibitors of activated STAT (PIAS) [66]. PIAS3 was found to block the DNA-binding activity of STAT3 and to inhibit STAT3-mediated gene activation. The regulation of PIAS is still unknown. However, more recent studies have identified other positive regula-tors for the JAK/STAT pathway that include serine kinases and interacting proteins [59].

STAT3 is linked to muscle atrophy Among the STAT genes, STAT3 has a critical role during development as evidenced by the early embryonic death of STAT3 knockout mice. In skeletal muscle, STAT3 has an important role in regulating myogenic differentiation in a context dependent manner, possibly because of the specific partners of the JAK/STAT pathway [67, 68]. Furthermore, several studies linked cancer cachexia with STAT3 activation through interleukin 6 (IL-6). IL-6 plays an important role in inducing cancer cachexia in mice bearing the colon-26 can-cer cell line and uterine cancer line, and the administration of IL-6 blocking agent reduces muscle wasting in these mice models [69-71]. IL-6 acts on cells by binding to IL-6 receptor, which leads to the activation of JAKs and later the activation of STAT3 and thus, up-regulates the acute phase response pro-tein gene expression in the liver [72, 73].

Cellular mechanisms of muscle atrophy Various pathophysiological conditions activate pathways that regulate protein turnover which leads to changes in muscle mass and performance. Atrophy is a decrease in the mass of a tissue or organ in response to cellular shrinkage caused by loss of organelles, cytoplasm, and proteins [74]. Several pathways for regulation of muscle atrophy in physiological and pathological conditions have been unrevealed, including the 2 proteolytic pathways that controls the

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protein turnover in muscle; the ubiquitin proteasome system and the autoph-agy lysosome system.

The ubiquitin proteasome system (UPP) Ubiquitination is a posttranslational modification where ubiquitin, a small reg-ulator protein (8.5 kDa), is attached to a substrate protein in a 3-steps process. In the first step, ubiquitin is activated by ubiquitin activating enzyme (E1) in an ATP dependent manner. The second step is mediated by ubiquitin conju-gating enzymes (E2) that transfer ubiquitin from E1 to E2, while the final step involves creating isopeptide bond between the substrate and ubiquitin and it is mediated by ubiquitin ligases (E3). After tagging the required substrate with ubiquitin, 26S proteasome targets the substrate for degradation. The 26S pro-teasome contains one 20S core subunit and two 19S regulatory subunits. After ubiquitination, the protein is recognized by the 19S subunits and later, the pro-tein is deubiquitinated, unfolded in an ATP dependent manner, and enters into the 20S subunit for degradation into short peptide fragments.

The rate limiting step in ubiquitination process is catalyzed by E3 ligase enzymes [75]. The human genome encodes more than 650 ubiquitin ligases [76] that are involved in the precise regulation of different cellular processes. Different E2-E3 pairs degrade different proteins, and the spasticity of E3s for certain protein groups provides selectivity to the degradation [75]. Among the known ligases, only a few are muscle specific and up-regulated during muscle loss [77]. Atrogin-1 and muscle RING finger-1 (MuRF1) are muscle specific E3 ligases that are up-regulated in skeletal muscle after atrophy induced con-ditions [78]. Several studies in muscle showed that MuRF1 is involved in the degradation of actin [79], troponin I [80], and thick filament proteins like my-osin heavy chain (MyHC), myosin binding protein C (MyBP-C), and myosin light chains 1 and 2 (MyLC1, and MyLC2) [81, 82]. In addition, up-regulation of Atrogin-1 has been linked to increased degradation of the myogenic regu-latory factor MyoD [83], MyHC, and the intermediate filament proteins vi-mentin and desmin [84]. Recently a group of novel E3 ligases that are regu-lated by Forkhead box class O family (FoxO family) transcription factor have been linked to muscle atrophy. Examples of these novel E3 ligases include Fbxo21 or SMART (specific of muscle atrophy and regulated by transcription) and Fbxo31. The function of these E3s is still unknown, but they were up-regulated in muscles in response to denervation induced atrophy [85].

Autophagy lysosomal pathway

Autophagy is a highly conserved pathway that degrades cytoplasmic material and organelles [86, 87]. This pathway is activated by stressful conditions such as fasting [88], viral infections [89], during aging [90], critical illness [91], and muscle denervation disuse [92, 93]. Autophagy can proceed in 3 different

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routes based on the route of delivery of content to the lysosomes. The first route is called macrophagy where a portion of the cytoplasm is wrapped inside autophagosome that later fuses with the lysosome. Another route of autophagy is the microphagy where the lysosome membrane sequester a portion of the cytoplasm [94]. The last route of autophagy is the chaperone mediated autoph-agy where proteins having a specific sequence signals are transported from the cytoplasm into the lysosome through lysosomal membranes [85].

Initially autophagy was considered a non-selective degradation pathway, but recent studies confirmed the presence of selective forms of autophagy like the selective removal of mitochondria. When mitochondria are damaged due to stress or other conditions, they are selectively degraded by autophagy in a process called mitophagy. The most characterized pathway for mitophagy is the PTEN-induced putative kinase 1 (PINK1) and Parkin pathway. PINK1 contains a mitochondrial targeting sequence and it is recruited into the mito-chondria. When PINK1 is imported into the inner membranes, it is rapidly cleaved and degraded, and thus the levels of PINK1 in normal cells are low [95]. In unhealthy mitochondria, the membrane potential becomes depolarized disrupting the inner membrane protein import, and thus PINK1 is no longer imported into the inner membrane. This leads to an increase in the concentra-tion of PINK1 in the outer mitochondrial membrane [96], and to recruitment of Parkin into the outer membrane. Parkin is a cytosolic E3 ubiquitin ligase, that starts ubiquitinating proteins on the mitochondrial surface, and this in turn attracts mitophagy inducing factors [97].

Another mechanism for mitophagy is through BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3), a pro-apoptotic mitochondrial pro-tein [98]. When the expression of Bnip3 is increased such as in hypoxia or starvation, the level of mitophagy increases [88, 99].

Inflammatory cytokines Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is a family of five transcription factors (p65, Rel B, c-Rel, p52, and p50) that me-diates several cellular processes such as apoptosis, immunity and inflamma-tion, and development and differentiation [100]. All NF-kB members are ex-pressed in skeletal muscles, and they are activated by inflammatory cytokines particularly tumor necrosis factor alpha (TNF-α) [75]. Despite the fact that NF-kB has been implicated in muscle atrophy caused by disuse and cachexia, the specific family members involved in the two types of atrophy are distinct [101]. Under normal conditions, NF-kB is maintained in its inactive state in the cytoplasm by binding to a family of inhibitory proteins called IkB. If TNF-α levels increased, the IkB gets phosphorylated by IkB kinase (IKKB) result-ing in ubiquitination and degradation of IkB. This fact releases NF-kB which then moves into the nucleus and activates gene transcription [102].

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Inflammatory cytokines can induce atrophy in several ways, one of these ways is the transcriptional activation of ubiquitination proteins by NF-kB [103]. For instance, over expression of IKKB in a muscle of transgenic mice showed severe muscle wasting that is mediated, at least in part, by MuRF1 but not by Atrogin-1 [104]. Furthermore, a recent study showed that TNF-α treat-ment caused up-regulation of myogenin, MuRF1 and Atrogin-1 [105].

Furthermore, both animal and clinical studies in cancer cachexia showed that the proinflammatory cytokines as TNF-α, IFN-γ, and IL-6 act as catalytic mediators in muscle atrophy associated with cachexia [106-108]. Recently, a study showed that TNF-α and IFN-γ can activate JAK kinases and therefore activate STAT3 in the cytoplasm of skeletal muscles. Later, the activated STAT3 forms a complex with NF-kB and the complex is rapidly imported into the nucleus where it binds to the promotor of iNOS gene, activating the tran-scription of iNOS/NO pathway, a known downstream effector of muscle loss induced by TNF-α and IFN-γ [109].

On the other hand, human studies showed that systemic administration of recombinant human IL-6 for 3 hours caused a 50% decrease in muscle protein turnover, with protein synthesis suppressed more than protein degradation [110]. In addition, systemic administration of different doses of IL-6 to rats for 7 days showed that only supraphysiological (high) doses of IL-6 was able to induce atrophy in the absence of any other disease [111, 112]. Further stud-ies showed that IL-6 can stimulate JAK/STAT3 pathway in several different pathways. The first pathway is through the up-regulation of the negative mus-cle regulator, myostatin [113]. Later, myostatin binds to activin type IIB re-ceptor and activate ALK4 and ALK5 which decreases the phosphorylation of S6 kinase and thus promotes muscle atrophy [114]. Other pathways by which IL-6 can induce muscle atrophy is through increasing the expression and acti-vation of caspase 3, and increasing the expression of CCAAT/enhancer-bind-ing protein delta (C/EBPδ) which later increases the expression of MuRF1 and Atrogin-1[115].

Apoptosis Apoptosis is an important cellular process in multicellular organisms that is highly cell specific and can be activated during both physiological and patho-logical conditions. Apoptosis is required for the survival of the organism and it is needed for maintenance of cell homeostasis, normal embryogenic devel-opment, development and function of the immune system, and for physiolog-ical cell turnover [116]. Mitochondria modulate and respond to cellular dy-namics through apoptotic signaling, which is activated when either OxPhos or their redox potential is disrupted, or in response to proapoptotic signals [117]. Mitochondria can induce apoptosis through either caspase-dependent or caspase-independent signaling mechanisms. Caspase-dependent signaling de-pends on the release of cytochrome c from the mitochondria, which triggers a

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cascade of actions by caspases that results in apoptosis. Briefly, cytochrome c and other proapoptotic factors are released from the mitochondria. Once re-leased, cytochrome c joins with apoptotic protease activating factor-1 (Apaf-1) and procaspase-9 to form a complex called the apoptosome. The apopto-some formation is followed by activation of several compounds that leads to degradation of DNA and initiation of cellular degradation. In the caspase-in-dependent pathway, the mitochondria release apoptosis-inducing factor (AIF) and endonuclease G (EndoG), which then induce chromatin condensation and DNA fragmentation. These pathways are regulated by a balance of proapop-totic (e.g., Bax) and anti-apoptotic proteins (e.g., Bcl-2), and by the mitochon-drial permeability transition pore (mPTP) [118].

Muscle wasting in intensive care unit Intensive care units (ICUs) have undergone several developments in the recent years leading to improved survival. Despite the fact that ICU interventions are lifesaving, they are associated with complications and negative consequences for morbidity and mortality. Muscle wasting and impaired muscle functions are common consequences of critical illness that leads to ICU patients wean-ing difficulties from the ventilator, increased duration of their hospital stay and reduced quality of patients’ lives [119, 120]. Regardless of the underlying causes, the muscle weakness is caused by the ICU condition [121, 122]. The clinical features associated with ICU acquired weakness are usually weakness of respiratory and limb muscles [123]. Respiratory muscles weakness is the main cause of prolonged duration of mechanical ventilation and difficulty in weaning from the ventilator [124], while weakness of the peripheral muscles can result in functional impairment, a reduced exercise ability and a reduced quality of life [125-127].

Critical Illness Myopathy (CIM) Critical illness myopathy (CIM) is a generalized progressive muscle weakness and atrophy mainly affecting limb and trunk muscles. Although patients with CIM have a reduced muscle excitability, their nerve conduction velocity re-mains intact. CIM is characterized by a specific loss of myosin and myosin-associated proteins, disorganized sarcomeres, unbalanced muscle metabolism, and altered protein turnover [119]. Several triggering factors have been sug-gested for CIM such as sepsis, multiple organ failure, systemic inflammation response, prolonged mechanical ventilation, corticosteroid and neuromuscular blockers, and unloading of muscles. Regardless of the underlying cause, CIM can complicate the course of the ICU patients’ treatment.

Despite several studies on CIM, the basic mechanism underlying CIM in the clinical setting is still poorly understood. This is partly caused by the fact

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that the CIM is usually complicated by the coexistence of more than one factor underlying muscle wasting. Different primary diseases, large differences in treatment, and the collection of muscle samples several weeks after admission to the ICU are factors that complicates the studies of CIM in the clinical set-ting. There is accordingly a compelling need for experimental models. In a series of studies performed on a novel ICU rat model that allowed to study muscle wasting under ICU modelled conditions without the presence of other compelling factors, Lars Larsson group showed that the cross sectional area (CSA) and specific force of single muscle fibers from the soleus and the EDL muscles were significantly reduced after 5 days of exposure to the ICU con-dition. In addition, mRNA expression of different isoforms of myosin and ac-tin in gastrocnemius muscle were reduced after 5 days of exposure to the ICU condition, while the myosin:actin ratio in the soleus and EDL muscles was reduced at 5 days and longer durations of exposure to the ICU condition. Fur-thermore, in the same rat model, the E3 ligases MuRF1 and Atrogin-1 were up-regulated from 6 hours to 8 days of exposure to the ICU condition [128].

Ventilator-Induced Diaphragm Dysfunction Ventilator-induced diaphragm dysfunction (VIDD) is a time dependent de-crease in diaphragm strength after initiation of mechanical ventilation, and it is the major cause of prolonged ventilator dependence in ICU patients [129-131]. Controlled mechanical ventilation (CMV) is an important factor in the development of VIDD, and the level of dysfunction progresses as the duration of CMV increases [131-133]. Current data indicates that the pathogenesis of VIDD includes both a reduction in force generation of the diaphragm and a rapid severe atrophy of muscle fibers [131, 134-139]. However, a recent study on an ICU rat model showed that the loss of diaphragm function starts only after 6 hours of exposure to the ICU condition, while the diaphragmatic atro-phy starts after 5 days of exposure to the ICU condition indicating that the loss of diaphragm function precedes its atrophy [139].

The underlying mechanism of VIDD is still unclear, but several studies linked VIDD to increased protein degradation via ubiquitin-proteasome path-way [140, 141], and activation of calpain and caspase 3 [134, 139, 142]. In addition, CMV is associated with oxidative stress in the diaphragm, as evi-denced by an increase in protein carbonylation between 6 hours and 8 days of exposure to CMV [139], and as evidenced by an increase in lipid peroxidation in the diaphragm in response to CMV [143]. Furthermore, mass spectrometric analysis of myosin isolated from diaphragm muscles of rats exposed to CMV for different durations, showed an increase in posttranslational modification in the myosin molecules especially in the rod region [139].

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Therapeutic approaches Several therapeutic strategies was proposed to prevent muscle wasting asso-ciated with the exposure to the ICU condition such as antioxidant therapy, nutritional interventions, and the use of growth hormone and testosterone de-rivatives [144-146]. However, none of these interventions improved the mus-cle function after exposure to the ICU condition [147]. Up to date, data on effective strategies to prevent and/or treat muscle weakness in critical illness remain limited, therefore further research is clearly needed.

BGP-15 Heat shock proteins (HSPs) are ubiquitous proteins that are found in all or-ganisms from bacteria to humans under both normal and stressful conditions like heat and oxidative stress [148]. HSP70 is a highly studied family of chap-erons that under normal conditions helps in folding proteins, while under stressful conditions prevents the aggregation of unfolded proteins and helps in their refolding [149]. Several studies have shown the importance of the induc-ible form of HSP70 under certain physiological and pathological conditions. Exercise or muscle injury increase the expression of HSP70, while prolonged periods of muscle inactivity reduces its level [150-152].

Using a porcine animal model resembling ICU conditions, we showed pre-viously that the CSA and specific force of single muscle fibers from biceps femoris did not change in response to 5 days of exposure to the ICU condition and this was associated with up-regulation in HSP70. However, when corti-costeroids, sepsis, or a combination of both were introduced in to the animals for 5 days in addition to the ICU intervention, the specific force was reduced in the biceps femoris with a significant down-regulation of the HSP70 [153-155]. Moreover, other studies using the same porcine model and an ICU rat model showed that the masseter muscle was spared in response to exposure to the ICU condition and this sparing was associated with an up-regulation in HSP70 [154, 156]. All these studies supported the hypothesis that an induction in HSP70 provide a protection against masseter and limb muscle wasting and loss of function observed in response to the ICU condition.

The HSP72 co-inducer BGP-15 is a multitarget compound that improves insulin sensitivity, increases mitochondrial volume, and improves metabolic homeostasis in several rat models of diabetes [157, 158]. It was recently shown that BGP-15 improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in the Duchenne's muscular dystrophy mice (mdx) model [159].

Muscle Passive loading Under normal conditions, skeletal muscle converts the mechanical stimuli into cellular responses that involve the regulation of several genes expression and biochemical events [160, 161]. Mechanical silencing, the loss of mechanical

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stimulation, in skeletal muscle is an important factor triggering the CIM phe-notype [162]. Since patient immobilization is indispensable in ICU patients under mechanical ventilation, passive mechanical loading of limb muscles was used in several studies to detect the effect of mechanical loading on CIM pathogenesis [162, 163].

Using an experimental ICU rat model, passive mechanical loading de-creased the loss in limb muscle mass and force generation associated with the ICU intervention. The improved maintenance of muscle mass and function in response to loading was associated with a reduced myosin loss, and decreased oxidative stress as evidenced by a decrease in protein carbonylation in the loaded limb [163]. In addition, the application of unilateral passive mechani-cal loading on the limb of deeply sedated and mechanically ventilated ICU patients showed that specific force of the fibers isolated from tibialis anterior was higher in the loaded than the unloaded side [162]. However, further in-vestigations are required to detect the underlying mechanism for the possible beneficial effects of mechanical loading during the exposure to the ICU con-dition.

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Aims of the thesis

The aim of this thesis is to investigate the possible beneficial effect of several intervention strategies and rehabilitation programs on the muscle wasting as-sociated with exposure to the ICU condition, and to detect muscle specific differences in response to exposure to the ICU condition and different inter-ventions in order to reach a conclusion on the possibility of the use of these intervention strategies in the clinic. In order to investigate these goals, a novel ICU rat model that allowed for deep sedation, mechanical ventilation and mus-cle unloading for duration ranging between 6 hours and 14 days was used.

Specific aims:

1. Investigate the effect of a novel HSP72 co-inducer, BGP-15, on the

diaphragm and soleus muscles for rats exposed to different durations of the ICU condition. Several molecular mechanisms for the potential effects of this drug were also evaluated with a great focus on the effect of this drug on posttranslational modification, protein synthesis and degradation pathways, and mitochondria related biomarkers. (Paper I, and II).

2. Unravel the mechanisms underlying the muscle sparing effect of me-

chanical loading during exposure to the ICU condition (Paper III). 3. Determine muscle-type specific differences in the activation of

JAK/STAT pathway in response to exposure to different durations of the ICU condition in order to develop specific intervention strategies associated with this pathway (Paper IV).

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Material and Methods

Animals (Papers I, II, III and IV) Female adult Sprague Dawley rats (SD) were used in all the experiments per-formed. With the exception of control, all experimental rats were mechani-cally ventilated, sedated with isoflurane and pharmacologically paralyzed with alpha cobratoxin. All animals were maintained in a protein and fluid bal-ance for the whole duration of the experiments which ranged from 6 hours to 14 days. The rats in the control group were sham operated, and thus they were sedated with isoflurane and received intra-arterial fluid but spontaneously breathing. The control rats were sacrificed 2 hours after the initial isoflurane anesthesia.

In papers I and II, the experimental rats that were treated with the chaperone co-inducer BGP-15, received a daily intravenous infusion of 40 mg/kg of BGP-15. However, for the rats used in the mechanical loading paper (paper III), the left leg was activated by a mechanical lever arm for 6 hours, at shortest duration, and 12 hours per day, for animals with 12 hours or longer durations. The mechanical lever arm produced a continuous passive ankle joint flexion and extension at a speed of 13.3 cycles/min.

Muscle biopsy (Papers I, II, III and IV) The soleus, extensor digitorum longus (EDL), tibialis anterior (TA), plantaris, and gastrocnemius muscles from both left and right legs, the intercostal, and diaphragm samples were dissected from the rats immediately after the animals were killed. Parts of these muscles were immediately frozen with liquid pro-pane and stored at -160oC. Parts of the diaphragm, EDL, and soleus were placed in relaxing solution at 4oC to dissect bundles of ~50 fibers per bundle for contractile measurement. A small part of the diaphragm and soleus were fixed in a 2.5% glutaraldehyde in 0.1 M cacodylic acid buffer at pH 7.4, and incubated at 4oC for at least 48 hours for subsequent TEM analysis.

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Membrane permeabilization and single muscle fiber contractile measurements (papers I and II) After collecting the bundles of muscle fibers from the required muscle, the bundles are treated with skinning solution for 24 hours at 4oC, and then they were transferred into -20oC. Within 1-2 weeks, the bundles were treated with cryo-protectant, frozen in liquid propane, and then stored at -160oC. On the day of the contractile measurement, the cryo-protectant was removed prior to contractile measurements and a single muscle fiber segment was removed from the muscle bundle and attached to two connectors in the single fiber set-up. Fiber CSA, maximum force and maximum force normalized to CSA (spe-cific force) were measured at the single muscle fiber level [164, 165].

Modified single fiber myosin in vitro motility assay for speed and force measurements (papers I and II) Unregulated actin was purified from rabbit skeletal muscle [166] and fluores-cently labelled with rhodamine–phalloidin (Invitrogen, Carlsbad, CA, USA). Myosin was extracted from rats’ single fibers and subsequently tested. The single-fiber myosin in vitro motility system has been extensively described in detail elsewhere especially the speed and force analyses [167]. Briefly, for speed analysis, from each single fiber preparation, 20 actin filaments that were moving at a constant speed in an oriented motion were selected. A filament was tracked from the center of mass, and the speed was calculated from 10 frames at an acquisition rate of five or one frame(s) per second, depending on the fiber type, and then the mean speed of the 20 filaments was calculated. For force analysis, the regression line was plotted between the relative fraction of moving filaments and alpha-actinin concentration, i.e., the number of moving filaments normalized to moving filaments prior to addition of alpha-actinin versus increasing alpha-actinin concentrations, and the x-axis intercept was used as an index to evaluate the force generating capacity of myosin.

Transmission electron microscopy (papers I and II) After fixing in glutaraldehyde solution, the samples were post fixed with 1% osmium tetraoxide for 3 hours at room temperature, and then dehydrated with increasing concentration of ethanol (70, 95, and 99%, three times each). Later the samples were infiltrated with propylene oxide for 10 min, and then infil-trated with increasing concentration (50 and 100%) of agar resin (agar 100 resin, agar scientific, UK) in propylene oxide over 24 hours. Later, the samples were placed in the desired molds with 100% resin and incubated at 60oC for 3

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days. To insure the quality and the orientation of the ultrathin sections, the samples were first trimmed and semithin sections were obtained, stained with toluidine blue, and viewed using a light microscope. Ultrathin sections of 60-80 nm were cut using the diamond knife of an ultramicrotome. Sections were then transferred into cupper grids and stained with uranyl acetate and lead cit-rate. Finally, the sections examined with transmission electron microscope (JEOL 1230; JEOL Ltd., Tokyo, Japan) operated at 100 kV, and digital mi-crographs were taken using Gatan multiscan (model 791, Gatan, Pleasanton, CA) using Gatan digital software version 3.6.4 (Gatan, Pleasanton, CA).

To analyze both IMF and SS mitochondria, 10 electron micrographs for 5 random fibers in the longitudinal orientation were obtained at 10.000x magni-fication. From each fiber, 2 random images for IMF and SS mitochondria ware captured, allowing the analysis of ≈40 IMF and 25 SS mitochondria. For each rat, a minimum of 200 IMF and 125 SS mitochondria were analyzed. Each electron micrograph was then analyzed using Image J (1.48 version, national institute of health, Bethesda, MD) by manually tracing outlines of IMF and SS mitochondria [168], and then taking the ratio between the normal and ab-normal mitochondrial morphology for each micrograph.

Respiratory chain enzymatic activity (paper I) Twenty mg of frozen diaphragm muscles were dissected into small fragments using a scalpel blade. Then the samples are diluted as 1:20 in ice cold sucrose muscle homogenization buffer (250 mM) in a 1-ml glass-glass tissue grinder. Later, the muscles were homogenized with 15 slow and controlled up and down strokes at 500 r.p.m. The homogenates were then centrifuged at 600 g for 10 min at 4oC, and the supernatants were used to measure the respiratory chain complexes and citrate synthase activities by spectroscopy [169, 170].

Complex I (NADH-ubiquinone oxidoreductase, EC 1.6.5.3) activity was measured by recording the decrease in absorbance of NADH at 340 nm (ε = 6.2/ mM.cm). The reaction was started by the addition of 6 µl of 10 mM ubiq-uinone to a cuvette containing the desired sample in a suitable solution (po-tassium sulfate buffer (0.05 M, pH=7.5), fatty acid free bovine saline albumin (BSA, 0.5 mg/ml), potassium cyanide (KCN, 0.3 mM), and NADH (0.1 mM)). Using spectroscopy, the decrease in absorbance of ubiquinone was monitored for 3 min. Specificity of complex I activity was measured by the addition of 10 μM of the complex I inhibitor, rotenone.

Complex II (succinate dehydrogenase; EC 1.3.5.1) activity was measured by recording the reduction of 2,6-dichlorophenolindophenol at 600 nm (ε = 19.1/ mM.cm). In a 1 ml cuvette, the sample was added to a solution contain-ing potassium sulfate buffer (0.025 M, pH=7.5), fatty acid free BSA (0.1 mg/ml), KCN (0.3 mM), succinate (20 mM), and 2,6-Dichlorophenolindophe-nol (2.175*10-3 %; Sigma, cat. no. 33125). The reaction was started by the

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addition of 4 µl of 12.5 mM decylubiquinone, and the decrease in absorbance was monitored for 3 min.

Complex III (ubiquinol cytochrome c oxidoreductase, EC 1.10.2.2) activ-ity was measured by measuring the reduction of cytochrome C at 550 nm (ε = 18.5/ mM.cm). The sample was added to a 1 ml cuvette containing potassium sulfate buffer (0.025 M, pH=7.5), 75 µm of oxidized cytochrome C, KCN (0.5 mM), Ethylenediaminetetraacetic acid (EDTA, 0.01 mM, pH=7.5), and tween-20 (0.025%). The assay was started by the addition of 10 µl of 10 mM decylubiquinol, and the increase in absorbance was monitored for 3 min. Spe-cific complex III activity was calculated by subtracting enzymatic reaction rates measured in parallel reactions with antimycin A (10 μg/ml).

Complex IV (cytochrome c oxidase; EC 1.9.3.1) activity was measured by recording the oxidation of the reduced form of cytochrome C at 550 nm (ε = 18.5/ mM.cm). In a 1 ml cuvette, the sample was added to a solution that con-tains potassium sulfate buffer (50 mM, pH=7.5), and reduced cytochrome C (0.06 mM). The reaction was started by the addition of 5 µl of muscle homog-enate and the decrease in absorbance was monitored for 3 min.

Complex II+III activity (succinate cytochrome C reductase) was measured by following the increase in absorbance of oxidized cytochrome C at 550nm (ε = 18.5/ mM.cm). In a 1 ml cuvette, the sample was added to a solution containing potassium sulfate buffer (0.2 M, pH=7.5), KCN (0.3 mM), and succinate (10 mM). The reaction was started by the addition of 50 µl of 1 mM oxidized cytochrome C and the absorbance was recorded for 3 min at 550 nm.

The activities of all the complexes for each sample were normalized to cit-rate synthase activity. Citrate synthase activity was measured by following the increase in the absorbance of oxaloacetic acid at 412 nm. In a 1 ml cuvette, the sample was added to a solution that contains Tris (100 mM, pH=8) with triton X-100 (0.2%), 100 µl 2,2'-Dithiobis 5-nitropyridine (DTNB; Sigma, cat. no. D218200), and acetyl coenzyme A (0.3 mM). The reaction was followed for 3 min after the addition of 50 µl of 10 mM oxaloacetic acid.

Lipidomics, proteomics, and mass spectroscopy (Paper I) To detect the levels of cardiolipin, Lipids were extracted from the diaphragm samples, and mass spectrometric analysis equipped with robotic nanoflow ion source was performed according to the method of Antal et al. [171].

Mass spectroscopy was used to detect posttranslational myosin modifica-tions. The slow and fast myosin isoforms were separated from diaphragm ly-sates using 6% SDS-PAGE gel. The bands were then excised and proteins were in-gel digested. The digests were then separated on a reverse phase C18 nanocolumn using Thermo Scientific EASY-nLC 1000 system. Elution is oc-curred by gradient increase of acetonitrile from 5% to 50% in 25 min at flow rate of 0.2 μL/min. MS/MS analysis of eluted peptides was performed using

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online-coupled Q Exactive Plus Orbitrap mass spectrometer (Thermo Scien-tific). Survey and data-dependent scan were recorded at 70,000 and 17,500 resolving power correspondingly. Recorded spectra were analyzed by Prote-ome Discoverer 1.4 software (Thermo Scientific) with allowance of a set of variable peptide modifications.

Subcellular fractioning (paper III) Around 50 mg of gastrocnemius muscles were weighed and placed in a 1:5 ratio in ice cold homogenization buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, 0.2% albumin, and 0.01% protease inhibitor cock-tail, pH=7.4). The samples were later homogenized for 1 min at 1600 r.p.m using Teflon pestles. Later, the samples were centrifuged at 1000 g for 10 min at 4oC. At the end of centrifugation, the pellets represents cellular debris and nucleus. The supernatant were centrifuged again at 14000 g for 15 min at 4oC. After this centrifugation, the supernatant represents mitochondria-free cyto-sol, and the cytosol is aliquoted and stores at -80oC. The mitochondrial pellets were resuspended in a washing buffer that contains 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, and 1 mM EDTA. The solution is then centrifuged at 14000 g for 15 min at 4oC. The washing was repeated 3 times, and finally the mitochondrial pellets were resuspended with 30 µl of Ripa buffer and stored at -80oC for later use [172]. Prior to loading, samples were boiled for 5 min at 95oC in Laemmli buffer (161-0747, BIO-RAD, USA) with 10% β-mercap-toethanol. For both cytoplasmic and mitochondrial fraction, Western blotting was performed to detect the levels of cytoplasmic and mitochondrial AIF and cytochrome C.

Western blotting

SDS-PAGE (I, III and IV) In paper I, Western blotting was used to detect the levels of MFN1, MFN2, OPA1, DRP1, HSP72, HSP90, αβ-crystalline, phosphorylated and unphos-phorylated insulin like growth factor 1 receptor (IGF1R), and total and cleaved poly (ADP-ribose) polymerase (PARP). In paper III, Western blotting was used to detect the levels of cytosolic and mitochondrial cytochrome C and AIF, and the levels of caspase 3 in the gastrocnemius muscle. While in paper IV, Western blotting was used to detect the phosphorylated and total levels of JAK1, JAK2, and STAT3 in the plantaris, intercostals and diaphragm. To de-tect the levels of these proteins, muscle lysates were loaded into a staking gel with 4% acrylamide concentration, and a running gel of 12% acrylamide. Electrophoresis was performed at 120 volt for 90 min, and then transferred to

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polyvinylidene fluoride (PVDF) membranes for Western blotting. After trans-fer, the membranes were incubated with the required primary antibody over-night at 4oC. The immunoblots were then incubated with secondary antibodies for 1 hour at room temperature, and scanned by Odyssey Imaging System (LI-COR Biosciences, UK). The signal intensities of each protein was quantified using the volume integration function (arbitrary unit), and the values were normalized to actin, actinin or complex IV.

Blue native (paper I) To detect the association of individual respiratory chain complexes into su-percomplexes, mitochondrial pellets were prepared from diaphragm muscle samples after homogenization in mitochondria isolation buffer using Teflon pestles operated at 1600 rpm. After homogenization, the samples were centri-fuged at 1000g for 10 min at 4oC to remove cellular debris and nuclei. The supernatant was centrifuged again at 14000 g for 15 min at 4oC. At the end, the mitochondrial pellets were resuspended with cold 1X Native Page (Invi-trogen) sample buffer containing 4% digitonin and centrifuged at 16000g for 20 min. The supernatant was collected, and 1µl of Native Page 5% G-250 Sample Additive was added. Later, equal amounts of mitochondrial protein were loaded into 3-12% Bis-Tris gel, Native PageTM Novex (Invitrogen), separated according to the manufacturer protocol (Native PageTM Bis-Tris gel, Invitrogen), and then transferred to polyvinylidene fluoride (PVDF) mem-branes for Western blotting and processed as previously described.

To detect supercomplexes, primary antibodies against NDUFA8 subunit of complex I, core 2 subunits of complex III, and COX I subunit of complex IV were used. The membranes were incubated with secondary antibody and pro-cessed using ECL Advance Western blotting detection kit according to man-ufacturer’s instructions. The immunoblots were subsequently scanned by Od-yssey Imaging System (LI-COR Biosciences, UK). The signal intensities of supercomplexes, and complexes I, III, and IV were quantified using the vol-ume integration function (arbitrary unit), and the ratios of supercomplexes SC to each of the complexes were calculated.

Gene expression analysis

Mitochondrial copy number (Paper I) For mitochondrial copy number measurement, the pellets obtained from ho-mogenization of diaphragm samples for respiratory chain complexes activities were used. DNA was isolated according to Gentra Puregene Tissue Kit (158667, QIAGEN, USA) manufacturer instructions. The final DNA pellets were resuspended in Tris-EDTA (TE) solution. DNA was amplified in tripli-

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cate using MyiQ single color real-time PCR detection system (Bio-RAD La-boratories, Inc.). The thermal cycling conditions include 95°C for 10 minutes, followed by 40 cycles of a PCR with denaturation at 95°C for 30 seconds and annealing and extension steps at 60°C for 30 seconds, 72°C for 1 minute, and 65°C for 10 seconds. Each reaction was performed in a 25 µl volume with 3 ng DNA, 1 µl of each primer, and 0.5 µl of SYBR Green. Primer sequences for D-loop mtDNA were: forward- GGGCCATCAATTGGTTCA, reversed- CTTCATGCCTTGACGGCTA., Primers were purchased from Sigma-Al-drich. The values were normalized against 18S (GenBank accession number AF102857), and the results were expressed as the ratio of the copy number of each group to the copy number of the control group.

RNA isolation (paper I, II and III) Total RNA was extracted from diaphragm (paper I), soleus (paper II), and plantaris (paper III) muscle tissues (10-30mg) using QiagenRNeasy Mini Kit (Qiagen, Inc.). Muscle tissues were first homogenized using a motor homog-enizer, and QIAshredder columns were used to disrupt DNA. Later, total RNA was eluted from RNeasy columns with 30 µl of RNase-free water. RNA con-centrations were later quantified using Nanodrop 1000 spectrophotometer.

Quantitative real-time Polymerase chain reaction (paper I, II and III) To measure the expression level of several mitochondrial dynamic and bio-genesis markers (paper I, II, and III), myosin heavy chain isoforms and actin (paper II), E3 ligases (paper II, and III), and mitophagy markers (paper III), qPCR was performed on reversed transcribed RNA isolated from the tissues mentioned above. cDNA was amplified in duplicate using MyiQ single color real-time PCR detection system (Bio-Rad Laboratories, Inc.). The thermal cy-cling conditions included 95oC for 10 min, followed by 50 cycles of a two-steps PCR with denaturation at 95oC for 15 seconds and a combined annealing and extension step at 60oC for 1 min. Threshold cycle (Ct) data obtained from running qPCR were related to a standard curve to obtain the starting quantity (SQ) of the template cDNA. The values were normalized to TBP or GAPDH.

RNA-seq data generation and read mapping (paper IV) Total RNA was extracted from plantaris, intercostal, and diaphragm muscles using MagMAX kit (Life tech, Carlsbad, CA, USA). mRNA was purified from 4 μg total RNA using Dynabeads mRNA Purification Kit (Invitrogen, Waltham, MA, USA). Strand-specific RNA-seq libraries were prepared using ScriptSeq mRNA-Seq Library Preparation Kit (Epicentre). Twelve-cycle PCR was performed to amplify libraries. Sequencing was performed on Illumina HiSeq2000 (Illumina) by multiplexed single-read run with 33 cycles. The overall read quality per sample was evaluated with FastQC to retain only sam-ples with sufficient quality. Subsequently, the reads were mapped to rat ge-nome (RGSC 5.0/rn5) using commercial software CLCBio with two allowed

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mismatches. For each gene, the reads mapped to the sense-strand exons of the gene were identified and counted.

Statistics (paper I, II, III and IV) Sigma Plot 13 software was used to perform statistical analysis. One-way analyses of variance (ANOVA) and the Tukey post hoc test were used when comparing multiple groups. If the analyzed data measures failed the normality testing, ANOVA on ranks, and Dunn’s post-hoc test were used (Paper I, II, and IV). To compare the effect of BGP-15 after 5, and 10 days of ICU inter-vention, unpaired student t-test was used (Paper I, and II). Linear regression was used to assess the correlation between changes in mitochondrial structure and duration of CMV. Fisher exact test and chi-squared comparison of the distributions were used to analyze the difference in the proportion of abnormal mitochondria between 10d and 10d+BGP-15 groups (Paper I). In addition, two way ANOVA and the Tukey post hoc test were used when comparing the effect of loading on different duration of mechanical silencing (paper III). P<0.05 was considered statistically significant.

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Results and Discussion

BGP-15 improves muscle fibers and myosin function following exposure to the ICU condition (paper I and II). Increasing the duration of exposure to mechanical ventilation leads to progres-sive loss of diaphragm muscle fibers specific force associated with a decrease in diaphragm muscle fiber CSA [173]. The administration of the chaperone co-inducer BGP-15 for 5 and 10 days of mechanical ventilation increased the specific force of the diaphragm muscle fibers without improving its CSA. To check if CMV and BGP-15 treatments affected myosin speed and force gen-eration, myosin molecules were isolated from diaphragm muscle of rats ex-posed to 10 days CMV, and rats exposed to10 days CMV with BGP-15 treat-ment. The results showed that both speed and force generation capacity of the myosin molecules isolated from diaphragm exposed to CMV were severely reduced, while BGP-15 treatment improved myosin force generation capacity.

In addition, in the soleus muscle, the specific force of the muscle fibers was reduced gradually after 5, 8 and 10 days exposure to the ICU condition and this was associated with reduced fiber CSA at all durations. However, BGP-15 administration for 5 days was able to increase the soleus fibers specific force and thus reverse the negative effects of exposure to the ICU condition for 5 days, while 8 and10 days of BGP-15 treatment failed to improve the soleus force generation. Furthermore, the loss of CSA in soleus after exposure to the ICU condition for 5, 8 and 10 days was not improved by BGP-15 treat-ment.

In order to investigate the inability of BGP-15 to improve the specific force of soleus fibers after 8 days, we analyzed the myosin:actin ratio in both soleus and diaphragm muscles after exposure to the ICU condition for 5, 8 and 10 days with and without BGP-15 administration. In accordance with our previ-ous findings [173, 174], the myosin:actin ratio in the diaphragm was not re-duced in response to the ICU condition, while the myosin:actin ratio was re-duced in the soleus muscle after 8 days of exposure to ICU condition regard-less of BGP-15 treatment. These results support our hypothesis that VIDD is caused by, at least in part, modifications within the contractile structures in the diaphragm, such as post-translational modification (PTMs) in myosin ra-ther than only increasing the catabolism of contractile proteins in the dia-phragm, while the hallmark of CIM in ICU patients in limb muscles is the preferential myosin loss. According to our results, BGP-15 was able to reduce

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the PTMs in the diaphragm, but was not able to alleviate the myosin loss after prolonged exposure to the ICU condition in soleus.

BGP-15 treatment reduced PTMs associated with exposure to the ICU condition (paper I) VIDD has been linked to increased oxidative stress in the diaphragm, and sev-eral studies showed that the administration of antioxidants to animals at the onset of mechanical ventilation prevents the development of VIDD [175, 176]. Using X-ray diffraction, previous studies on our ICU rat model, showed that the loss of diaphragm muscle function was associated with PTMs in the thick filament backbone [139]. In addition, LC/MS analysis of the myosin heavy chain showed an increase in the detected PTMs (deamidation, nitration, and oxidation) in the myosin heavy chain tail.

Our results in the diaphragm showed that BGP-15 administration for 10 days during exposure to the ICU condition decreased PTMs in the myosin heavy chain and decreased myosin SUMOylation. SUMOylation is a reversi-ble PTM that regulates the function of many proteins, and it is modulated by chemical and physical stressors. The reduced PTMs are suggested to be, at least in part, responsible for the positive effects of BGP15 on myosin force generation and diaphragm function following 10 days of CMV exposure.

BGP-15 has positive effects on mitochondria structure, dynamics, and biogenesis (paper I and II) Although the mechanisms underlying VIDD are still obscure, increased oxi-dative stress in the diaphragm is forwarded as a possible triggering factor of VIDD [177]. Several studies suggested that this increase in oxidative stress is induced by mitochondrial related ROS [52, 178, 179], and several mecha-nisms were tested to understand the link. The first mechanism suggests that prolonged muscle inactivity disturbs intracellular calcium balance and in-creases cytosolic levels of calcium within the inactive muscle fibers [180], which will likely lead to increased calcium uptake by mitochondria. Inside mitochondria, calcium enhances citric acid cycle, production of NADH, and enhances OxPhos activity, and thus, increases production of ROS [181, 182]. The second mechanism is that increased mitochondrial levels of fatty acid hy-droperoxides, which will then interact with components of OxPhos complexes [183]. An impaired protein import into mitochondria resulting in mitochondria dysfunction may also increase ROS production. The majority of mitochon-drial proteins are nuclear encoded, and mitochondrial biogenesis requires the protein import machinery to transport these cytosolic proteins into the mito-chondria [184].

In our study (paper I), the early exposure to CMV disrupted mitochondrial structure, resulting in more vesicular and swollen structures. The dynamic of mitochondria was also affected during CMV with lower levels of the mito-chondrial fusion gene, MFN1, and less levels of the fission protein, DRP1.

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Furthermore, mitochondrial function was impaired by reducing the activity of complex IV, and the mitochondrial content and biogenesis markers. However, in the diaphragm, 10 days of BGP-15 treatment improved mitochondrial func-tion by increasing the activity of the respiratory chain complexes III and IV. It also improved mitochondrial biogenesis and content by respectively in-creasing the gene expression of PGC-1α and increasing cardiolipin levels. However, BGP-15 did not reduce the negative effects caused by CMV on mi-tochondrial structure, although it increased mitochondrial dynamic by increas-ing the protein levels of MFN2, OPA1, and DRP1 (Figure 2).

Furthermore, in the soleus muscle (paper II), exposure to the ICU condi-tion for 5 and 10 days caused a change in the mitochondrial morphology, and a decrease in mitochondrial biogenesis. BGP-15 treatment for 10 days im-proved mitochondrial dynamics as evidenced by increasing the gene expres-sion of MFN1, MFN2, and DRP1. Moreover, BGP-15 treatment for 5 and 10 days improved mitochondrial structure and biogenesis (Figure 2).

The increase of PGC-1α by BGP-15 in both the diaphragm and the soleus muscles is important not only for increasing the mitochondrial mass in the muscle and thus increasing ATP supply to prevent bioenergetic crises, but also for improving mitochondria maintenance by continuously providing newly synthesized mitochondrial component [185]. In addition, mitochondrial maintenance is further improved by BGP-15 treatment through its effects on improving mitochondrial dynamics. As we showed in the soleus muscle, the improved mitochondrial maintenance would decrease the production of ROS and protect against mitochondrial structural damage. On the other hand, de-spite the positive effects of BGP-15 treatment on diaphragm mitochondria, the drug failed to improve the mitochondrial structure, indicating the presence of other players that are affecting mitochondria structure (Figure 2).

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Figure 2. The effects of exposure to the ICU condition, mechanical loading and BGP-15 treatment on mitochondria

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The effects of BGP-15 on transcriptional regulation of protein synthesis and degradation (paper II) Increased oxidative stress in the diaphragm can activate some proteolytic path-ways such as calpain and caspases, and some signaling pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)[186] and sig-nal transducer and activator of transcription (STAT) [129]. We showed before that both VIDD and CIM are associated with early activation in the UPP for protein degradation as evidenced by up-regulation in the E3 ligase MuRF1 in the diaphragm and limb muscles after 6 hours of exposure to the ICU condi-tion [173, 187].

In order to understand the differences in response to BGP-15 administra-tion between the soleus and the diaphragm muscles, we analysed the gene ex-pression of UPP for protein degradation and the gene expression of myosin and actin in both the diaphragm and soleus of rats exposed to the ICU condi-tion for 5 and 10 days with and without BGP-15 treatment. In the diaphragm, MuRF1 and Atrogin-1 expressions were induced only during 5 days of expo-sure to CMV, while the Fbxo31 levels were induced after both 5 and 10 days of CMV. BGP-15 administration for 5 days reduced the expression of Fbxo31 in the diaphragm. In addition, the gene expression of SMART, myosin heavy chain IIa and IIx, and actin were not influenced by CMV or BGP-15 treatment. On the other hand, in the soleus muscle, our results showed that the gene ex-pression of all the tested E3 ligases (MuRF1, Atrogin-1, SMART, and Fbxo31) were increased after 5 days exposure to the ICU condition, while the reduction in the expression of myosin on gene and protein levels were noticed after 10 days of exposure to the ICU condition. However, BGP-15 treatment showed a trend of reducing the gene expression of MuRF1, and was able to reduce the expression of Fbxo31 during exposure to the ICU condition (Fig-ure 3A).

These results indicate that the improved diaphragm function after 10 days exposure to the ICU condition and the improved soleus function after 5 days exposure to the ICU condition are not related to any positive effects of the drug on myosin expression or myosin breakdown but rather through a differ-ent mechanism, as discussed above, reduced PTMs. In accordance with this hypothesis, BGP-15 treatment reduced the overexpression of Fbxo31 in both diaphragm and soleus following exposure to the ICU condition, and the re-duced Fbxo31 is an indicator that BGP-15 administration is associated with reduced oxidative and genotoxic stress in both muscles. This is supported by a previous study that showed induced Fbxo31 levels in response to several genotoxic conditions like UV and X-ray irradiation, and oxidative stress in-duced by H2O2 or addition of the chemotherapeutic DNA damaging agent etoposide [188].

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Figure 3. The effects of exposure to the ICU condition, mechanical loading and BGP-15 treatment on protein degradation pathways in muscles: The ubiquitin pro-teasome system (A), Apoptosis (B) and JAK/STAT pathway (C).

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The chaperone co-inducer BGP-15 has a multifactorial mechanism of action (paper I) Despite its action as a HSP72 co-inducer, BGP-15 has several other targets like the phosphorylation of IGF1R [189] and the inhibition of PARP1 pathway [190]. Our results showed that 10 days of BGP-15 treatment during CMV ex-posure elevated the levels of HSP72, and prevented the over activation of PARP1 pathway. These results are of specific interest since PARP1 inhibition reduces mitochondrial ROS production and ROS-related oxidative damages during stress [191-193]. Moreover, previous studies on BGP-15 treatment on skeletal muscles showed that mitochondrial enzymes activities had a positive correlation with HSP72 mRNA expression [194]. HSPs also facilitate the im-port of nuclear encoded proteins into the mitochondria, and are thus crucial in maintaining the function of mitochondria [195]. Recently, BGP-15 was re-ported to have a significant effect on cardiac muscle in a mouse model of heart failure via insulin growth factor 1 receptor (IGF1R) phosphorylation which was independent of HSP expression [189], but in the present study, BGP-15 had no effect on phosphorylation of IGF1R.

Mechanical loading prevents the over expression of a novel set of E3 ligases (paper III) The ubiquitin E3 ligases MuRF1 and Atrogin-1 have been identified by sev-eral studies as genes that are induced in skeletal muscles by different atrophy inducible conditions such as immobilization, hind limb unloading, denerva-tion, cancer cachexia, and treatment with corticosteroids like dexamethasone [78]. As noted above, we previously noticed, using our ICU rat model, a dra-matic decrease in the force generating capacity (after 5 days of exposure to mechanical silencing) that was associated with an up-regulation in gene ex-pression of MuRF1 and Atrogin-1 in the limb muscle only after few hours of mechanical silencing [153]. In addition, when we applied mechanical loading to one of the rat hind limb, we noticed a sparing of muscle mass and function on the loaded side as evidenced by a higher muscle mass and higher specific force generation on the loaded side than the unloaded side of rats exposed to the ICU condition for 9-14 days [187]

Our results showed that mechanical silencing induced the gene expression of MuRF1 after 6 hours of exposure to mechanical silencing, while the gene expression of Atrogin-1, Fbxo31 and SMART increased following 5 days of exposure to mechanical silencing. Mechanical loading, on the other hand, counteracted the induction of the novel E3 ligases Fbxo31 and SMART with-out affecting MuRF1 and Atrogin-1. According to our results, the positive ef-fects of mechanical loading on the limb muscle was not mediated through MuRF1 or Atrogin-1, but rather was associated with reduced levels of the novel E3 ligases Fbxo31 and SMART (Figure 3A). This is of special interest

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since under mechanical silencing the levels of these novel E3 ligases was ele-vated after 5 days of exposure to the ICU condition which mirrors the time of preferential myosin loss and the loss of limb muscle function as we previously showed. Fbxo31, as previously described is usually up-regulated in response to oxidative or genotoxic events, and thus, the reduced Fbxo31 levels during mechanical loading indicated a less stress to the limb muscle. Furthermore, SMART showed to be important for inducing certain type of muscle atrophy as denervation while SMART inhibition protected against denervation atro-phy [85].

Mitochondria dynamics, biogenesis and mitophagy are affected by mechanical loading (paper III) The role of mitochondria in the regulation of signaling pathways that controls muscle atrophy is widely studied. The expression levels of mitochondrial fu-sion genes, Mfn1, Mfn2, and Opa1, and the fission gene, fis1 were signifi-cantly elevated after 5 days of mechanical silencing, while the fission gene Drp1 levels were down-regulated during 0.25-4d of exposure to mechanical silencing and up-regulated after 9 days of mechanical silencing. However, me-chanical loading was able to completely counteract the levels of all mitochon-drial markers that were affected by mechanical silencing (Figure 2). These findings indicate that mechanical silencing was associated with imbalance in mitochondrial dynamic that can affect the organelle and function and contrib-ute to muscle atrophy. This is supported by the fact that several studies have shown that mitochondrial dynamic imbalance contributes to muscle atrophy in response to denervation and fasting [196].

Furthermore, our study showed that the levels of the mitophagy markers, Pink1 and Parkin, were increased after 5 days of exposure to mechanical si-lencing indicating that mechanical silencing increases mitophagy. On the other hand, mechanical loading decreased the levels of these mitophagy mark-ers. All these results suggest that mitophagy is affected by mechano-transduc-tion pathways within the skeletal muscle tissue. In addition, the effect of mechano-transduction on mitochondrial biogenesis was evaluated in our study. Our results showed that total PGC-1α expression was reduced follow-ing 5 days of mechanical silencing, and mechanical loading had no effect of restoring these levels (Figure 2). However, when we looked at different isoforms of PGC-1α, we noticed that PGC-1α4 levels were improved during 0.25-4 days of exposure to mechanical loading.

To maintain proper energy homeostasis in the cells, damaged mitochon-dria, as a result of mitochondria depolarization that is caused by defects in the oxidative phosphorylation reactions, should be removed by mitophagy and re-placed for new mitochondria [185]. Furthermore, PGC-1α as discussed earlier, suppresses oxidative stress and thus leads to less damaged mitochondria and less mitophagy. However, in our model, 5 days mechanical silencing in the limb muscle reduced PGC-1α and elevated mitophagy markers, indicating that

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the total count of mitochondria should be reduced by this condition. While mechanical loading prevented the increase in mitophagy without enhancing PGC-1α levels.

Mechanical loading reduces the release of pre-apoptotic factors from mitochondria (paper III) Cytochrome C release from the mitochondria induces cellular caspase depend-ent apoptosis [197], while the release of AIF into the cytoplasm, which then moves into the nucleus resulting in chromatin condensation, induces cellular caspase independent apoptosis. The release of AIF further permeabilized the mitochondrial membrane leading to release of cytochrome C and more AIF [198, 199].

In our study, we investigated the levels of cytochrome C and AIF in both cytosolic and mitochondrial fractions. Cytochrome C levels in the mitochon-dria did not differ between the controls and rats exposed to different durations of mechanical silencing and loading. However, in the cytosolic fraction, a sig-nificant increase in cytochrome C levels was observed during 0.25-4 days of mechanical silencing. To check if this increase was associated with increased caspase 3 activation, we evaluated total and cleaved caspase 3 levels in tissue homogenates, and we noticed an elevation in active caspase 3 after 9 days of exposure to mechanical silencing. On the other hand, mechanical loading had no effect on cytosolic cytochrome C levels or caspase 3 activation, indicating that the release of cytochrome C from the mitochondria was more obvious in the unloaded limb muscle (Figure 3B).

A significant release of AIF into the cytoplasm was observed following 9-14 days of mechanical silencing, even though AIF was released from mito-chondria regardless of the loading conditions, indicating that the release of AIF was more pronounced in the unloaded limb muscles.

JAK2/STAT3 pathway is activated in different muscles after the exposure to the ICU condition (paper IV) JAK/STAT pathway is a signaling pathway that regulates myogenesis and muscle differentiation, inflammation and other immunity processes [200]. Our results showed that the exposure to the ICU condition for different durations changed the expression of different genes that are involved in the JAK/STAT pathway in the plantaris, intercostal and diaphragm muscles. Using Western blotting, in the plantaris, we observed an activation in the JAK1/STAT3 path-way during 1-4 days of exposure to the ICU condition, and the JAK2/STAT3 pathway during the same period and again after 9 days exposure to the ICU condition. Furthermore, in the intercostal, the JAK2/STAT3 pathway was ac-tivated during 1-4 days of exposure to the ICU condition. In the diaphragm, the JAK2/STAT3 pathway was activated after 1 day of exposure to ICU con-dition and stayed elevated even after 9 days exposure to ICU condition (Fig-ure 3C). STAT3 activation by ICU intervention is of special interest to us

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since several studies have shown that exposure to CMV causes activation in STAT3 in the diaphragm and increases the mitochondrial production of ROS, while inhibiting JAK and thus STAT3 during CMV reduces ROS generation and improves diaphragm muscle contractility [201, 202].

The activation of STAT3 have been widely studied in cancer cachexia, where systemic inflammation evidenced by an overexpression of Tumor Ne-crosis Factor alpha (TNF-α), Interferon gamma (IFN-γ), and IL-6 have been implicated in both animal models of cachexia and human experiments [106-108, 203]. A recent study, showed that TNF-α and IFN-γ can activate JAK kinases and later activate STAT3 that later induces the gene expression of myostatin, leading to muscle atrophy [109]. In our study, both TNF-α and IFN-γ protein levels in the plasma were not increased in response to exposure to different durations of the ICU condition. In addition, the downstream sig-nals for TNF-α and IFN-γ /STAT3 muscle atrophy (iNOS) gene expression was not affected by exposure to the ICU condition, indicating that this path-way is not activated under our modelled ICU condition.

However, IL-6 can also induce muscle atrophy in several pathways that include inducing the gene expression of myogenin [113], caspase 3, and C/EBPδ and thus MuRF1 and Atrogin-1 [115]. Our study showed that myo-statin gene expression were rather reduced in response to long-term exposure to the ICU condition, and that the gene expression of activin type IIB receptor (myostatin receptor) was also reduced in response to exposure to the ICU con-dition in the plantaris, intercostal and the diaphragm muscles. These results support our previous finding that myostatin expression was reduced in the gas-trocnemius muscle after 5 days exposure to the ICU condition [204]. Further-more, our study showed that the expression of caspase 3 was increased in the plantaris and intercostals in response to long duration exposure to the ICU condition. These results are in accordance with our previous finding in the gastrocnemius where caspase 3 was activated after 9 days of exposure to ICU condition [205], while caspase 3 was activated in the diaphragm during 5-8 days of exposure to CMV [139]. Our results also showed that the expression of C/EBPδ was induced in the plantaris, intercostal and diaphragm muscles in response to short-term exposure to the ICU condition, while the expression of both Atrogin-1 and MuRF1 was increased by short- and long-term of exposure to the ICU condition in these muscles except the diaphragm where Atrogin-1 levels were induced only after short-term exposure to the ICU condition. These results support our previous findings that Atrogin-1 and MuRF1 were up-regulated in the limb muscle after few hours of exposure to the ICU con-dition, and that in the diaphragm, MuRF1 levels were also induced after few hours of exposure to ICU condition [206]. Furthermore, a recent study from our group tried to use HDAC4 blocker to evaluate the role of myogenin-HDAC4 axis in inducing MuRF1 expression and muscle atrophy associated with ICU intervention. The results of the study showed that inhibiting HDAC4 did not prevent the increased expression of MuRF1 caused by exposure to the

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ICU condition in the gastrocnemius, indicating the presence of a different pathway for inducing MuRF1 expression. And thus JAK2/STAT3 pathway might offer a suitable explanation for the increased MuRF1 expression during exposure to the ICU condition.

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Conclusions

Despite being a lifesaving treatment in critically ill intensive care unit (ICU) patients, mechanical ventilation has negative effects on diaphragm muscle function. The effect of the chaperone co-inducer BGP-15 was investigated in an experimental rat ICU model. We showed that the loss in diaphragm func-tion preceded the loss in muscle mass and correlated with post-translational modifications of the motor protein myosin. Ten days BGP-15 treatment, on the other hand, improved myosin and diaphragm function, reversed protein SUMOylation and improved mitochondrial content and function through, at least in part, induction of HSP72 and PARP1 inhibition.

BGP-15 treatment was also investigated in the soleus muscle to test the possible beneficial effects of this drug during CIM. Our study showed that the exposure to the ICU condition resulted in a gradual decline in soleus muscle mass and function, while BGP-15 treatment for 5 days reversed the loss in soleus muscle function without reversing muscle atrophy. However, treatment with BGP-15 for 8 and 10 days did not prevent the loss in the soleus muscle mass or function. The difference in response of the BGP-15 treatment between diaphragm and soleus muscles is thought to be related to the differences in the characteristics of both VIDD and CIM. In VIDD, the myosin:actin ratio is not influenced by exposure to the ICU condition, while in CIM, the preferential loss of myosin is an important factor in the pathogenesis. BGP-15 did not im-prove the function of soleus muscle after 8 days exposure to the ICU condition due to its inability to reduce myosin loss in the soleus, while in the diaphragm it was able to reverse, at least in part, posttranslational modifications and thus improve its function.

In addition to the use of heat shock protein co-inducer as a treatment strat-egy for CIM, we previously showed that the mechano-transduction pathways are important triggering factors in the pathogenesis of CIM, and previous stud-ies from our group showed the positive effects of mechanical loading in im-proving limb muscle function during exposure to the ICU condition, however, the mechanism underlying this effect is still under investigation. In the current thesis, our studies showed that passive mechanical loading of the limb muscle reversed the imbalance in mitochondrial dynamic and reduced the levels of mitophagy genes that were induced by mechanical silencing. Furthermore, mechanical silencing resulted in an early increase in the cytosolic cytochrome C levels followed by an activation of caspase 3. While, the AIF released from

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mitochondria into the cytosol increases mitochondrial membrane permeability and causes additional release of cytochrome C and AIF.

The investigation of other pathways that can be activated during the expo-sure to the ICU condition showed that the JAK2/STAT3 pathway is differen-tially activated in the plantaris, intercostal and the diaphragm muscles in re-sponse to exposure to different durations of the ICU condition. All the inves-tigated muscles showed an early activation of the JAK2/STAT3 pathway start-ing from the exposure to the ICU condition for 1-4 days, however, after 5 days of exposure to the ICU condition, the differences in the activation of this path-way becomes more obvious due to the presence of different levels of inhibition in different muscles. Further studies are required with the use of JAK2/STAT3 inhibition to fully examine the role of this pathway in the pathogenesis of CIM and VIDD prior to the translation to clinical research. The prevention/thera-peutic strategies against CIM and VIDD should take into account the muscle specific differences in response to exposure to the ICU condition, in addition to the common pathogenic factors like perturbed mechanical stimuli and mechano-transduction processes and increased systemic inflammatory state.

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Acknowledgments

This work was carried out at the department of physiology and pharmacology at Karolinska Institute, Stockholm, Sweden and the clinical neurophysiology department at Uppsala University, Uppsala, Sweden, with collaborations at the department of Clinical Neuroscience, clinical neurophysiology, Karolin-ska Institutet, Stockholm, Sweden, the department of Chemistry, Uppsala Uni-versity, Uppsala, Sweden, the department of woman and health, Padova Uni-versity, Padova, Italy, Institute of biochemistry, Biological research center, Hungarian academy of sciences, Szeged, Hungary, the Pennsylvania State University College of Medicine, Hershey, PA, USA, Inserm U845, Université Paris Descartes, Paris, France, the Department of Molecular Virology, Immu-nology and Medical Genetics, the Ohio State University, OH, USA, Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Min-nesota, USA and Regeneron Pharmaceuticals, NY, USA.

I want to thank all the staff, colleagues and friends for their direct or indirect contributing to this work. I would especially like to convey my deepest grati-tude to:

My supervisor, Professor Lars Larsson, I am grateful for your guidance, val-uable advices and for all the opportunities you have given me. I’m indebted to your encouragements, inspiration and assistance from the preliminary to the concluding level, which enabled me to develop a good understanding and pas-sion for skeletal muscle research. My co-supervisors, Professor Jonas Bergquist and Professor Roland Flink, for sharing their knowledge and skills. Thank you for all the help and support.

The prefects and administrative staff at the department of physiology and pharmacology at Karolinska Institute, the clinical neurophysiology depart-ment at Uppsala University, and the clinical neurophysiology department at Karolinska Institutet for all the support and help. All the staff working at Erasmus Mundus program / Dunia beam project that is funded by European Union, and the staffs at the scholarship offices at Upp-sala University, Sweden, and AN-Najah National University, Palestine for all

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the help and support during the first 34 months of my PhD study that was funded by Erasmus Mundus program. Our present and past group members, Yvette Hedström, Dr. Nicola Cacciani, Dr. Mesihan Li, Dr. Monica Llano Diez, Dr. Stefano Gasteldello, Hazem Ak-kad, Dr. Rebeca Corpeno, Dr. Hanna Ogilvie, Arvind Venkat Namuduri, An-ders Backeus, Gabriel Heras Arribas, Leonardo Traini, and Ann-Marie Gus-tafson for your excellent guidance. Thank you for your friendship, for sharing your tremendous knowledge and experience and for your support that has al-ways been my source of inspiration.

My family, especially mom and dad, you have always been there for me, giving me the strength when I’m tired, and helping me to stand up and carry on whenever I fall. أحبكم و أهدي ثمرة تعبي لكم

My husband, Derar, for standing by me, holding me on and never let me fall. Your love and your support were guiding me through this tough road, I couldn’t have done this without you. And finally, to my Summer, for making everything in my life joyful, and for making the struggle easier. أعشقكم و أعشق كل لحظة أمضيتها معكم في هذه الرحلة الشاقة .... و في نهاية هذه الرحلة أهديكم أجمل

ما فيها و أعز الذكريات الى قـلبي ...

51

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1368

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-328596

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UPSALIENSISUPPSALA

2017