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1 CALPAIN REGULATES AKT AND NF PROLONGED MECHANICAL VENTILATION By WILLIAM BRADLEY NELSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLM ENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 William Bradley Nelson
3 To W.W., who is always on the job
4 ACKNOWLEDGMENTS First, I want to acknowledge and thank my mentor Dr. Scott Powers f or the opportunities and support he has given me. Without him, this would not be possible. I also want to recognize my wife, Cami for her faith selflessness and support in this endeavor. Similarly, I thank my beautiful ch ildren for being happy to see me after a long day and for their countless smiles. Importantly, I also extend my gratitude to my parents for providing me with the upbringing that made this a realistic opportunity. Lastly, I thank my lab mates for their friendship, help and generosity.
5 TA BLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 LITERATURE REVIEW ................................ ................................ .......................... 15 Ventilator Induced Diaphragm Dysfunction is Clinically Significant ........................ 15 Ventilator Induced Diaphragm Dysfunction Occurs Rapidly ................................ ... 16 Mechanical Ventilation Promotes Protein Turnover ................................ ................ 17 Mechanical Ventilation Activates Proteolytic Pathways ................................ .......... 18 Ubiquitin Proteasome Pathway (UPP) Mediated Proteolysis ........................... 18 Caspase 3 Mediated Proteolysis ................................ ................................ ...... 19 Calp ain Mediated Proteolysis ................................ ................................ ........... 20 Autophagy Lysosomal Pathway (ALP) Mediated Proteolysis ........................... 20 Calpain is an Important Regulatory Proteas e ................................ ......................... 22 Calpain as a Signaling Molecule in Skeletal Muscle ................................ ............... 24 Calpain Impacts Akt Signaling ................................ ................................ .......... 25 Calpain Can Activate NF ................................ ............................. 26 Summary ................................ ................................ ................................ ................ 26 3 METHODS ................................ ................................ ................................ .............. 29 Experimental Design ................................ ................................ ............................... 29 Control Animals ................................ ................................ ................................ ...... 29 Mechanically Ventilated Animals ................................ ................................ ............ 29 Calpain Inhibition ................................ ................................ ................................ .... 30 Western Blot Analysis ................................ ................................ ............................. 31 RNA Isolation and cDNA synthesis ................................ ................................ ......... 32 Real Time Polymerase Chain Reaction ................................ ................................ .. 32 TUNEL Analysis for Apoptosis ................................ ................................ ................ 32 20S Proteasome Activity A ssay ................................ ................................ .............. 33 In Vitro Analysis of Calpain Specificity ................................ ................................ .... 33 Trolox Equivalency Antioxidant Capacity Assay ................................ ..................... 34 Statistical Analysis ................................ ................................ ................................ .. 34 4 RESULTS ................................ ................................ ................................ ............... 35
6 Physiological Responses to Prolonged MV ................................ ............................ 35 Pharmacological Inhibition of Calpain Activity in the Diaphragm ............................ 35 Calpain Inhibition Maintains Akt Phosphorylation ................................ ................... 36 Calpain Inhibition Prevents Akt/FoxO3a Atrophy Signaling ................................ .... 36 Calpain Inhibition Prevents Apoptosis ................................ ................................ .... 37 Active Calpain Promotes 20S Proteasome Activity ................................ ................. 38 Evidence that SJA 6017 is a Selective Calpain Inhibitor ................................ ......... 38 C ................................ ....................... 39 Active Calpain Regulates NF ................................ ................................ 39 SJA 6017 Does Not Exhibit Antioxidant Capacity ................................ ................... 39 5 DISCUSSION ................................ ................................ ................................ ......... 58 Overview of the Principal Findings ................................ ................................ .......... 58 Calpain Activity Regulates Akt Phosphorylation ................................ ..................... 58 Calpain Activity Regulates Akt/FoxO3a Proteolytic Signaling ................................ 60 Active Calpa in Increases the Expression of Ubiquitin Ligases ......................... 60 Autophagy Gene Expression is Regulated by Active Calpain .......................... 61 MV Induced Calpa in Activation Regulates NF ................................ ...... 63 Critique of the Experimental Model ................................ ................................ ......... 65 Conclusions and Future Directions ................................ ................................ ......... 66 LIST OF REFERENCES ................................ ................................ ............................... 68 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 79
7 LIST OF FIGURES Figure page 3 1 Proposed calpain signaling pathway in the diaphragm during prolonged mechanical ventilation.. ................................ ................................ ...................... 28 4 1 Protein level of the active form of calpain 1 in diaphragm of experimental groups. ................................ ................................ ................................ ............... 41 4 2 II spectrin breakdown product (SBPD) in diaphragm of experimental groups.. ................................ ................................ ... 42 4 3 Protein levels of phosphorylated Akt in diaphragm of experimental groups.. ..... 43 4 4 Protein levels of total Akt in diaphragm of experimental groups.. ....................... 44 4 5 mRNA levels of FoxO3 in diaphragm of experimental groups.. .......................... 45 4 6 mRNA levels of MuRF1 in diaphragm of experimental groups.. ......................... 46 4 7 mRNA levels of atrogin 1 in diaphragm of experimental groups.. ....................... 47 4 8 mRNA levels of BNIP3 in diaphragm of experimental groups.. ........................... 48 4 9 mRNA levels of cathepsin L in diaphragm of experimental groups. .................... 49 4 10 mRNA levels of LC3 in diaphragm of experimental groups.. .............................. 50 4 11 Protein levels of MuRF1 in diaphragm of experimental groups.. ........................ 51 4 12 Protein levels of atrogin 1 in diaphragm of experimental groups. ....................... 52 4 13 TUNEL positive nuclei in diaphragm of experimental groups.. ........................... 53 4 14 Chymotrypsin like 20S protea some activity in diaphragm of experimental groups.. ................................ ................................ ................................ .............. 54 4 15 In vitro 20S proteasome activity.. ................................ ................................ ........ 55 4 16 n diaphragm of experimental groups. ........................... 5 6 4 17 p50 DNA binding activity in diaphragm of experimental groups.. ........................ 57
8 LIST OF ABBREVIATION S Akt Protein kinase B A LP Autophagy lysosomal pathway cm Centimeter cDNA Complementary deoxyribonucleic acid DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraaecitic acid F I O 2 Fraction of inspired oxygen FoxO Forked box g Gravity H 2 O Water Hg Mercury IC 50 Half maximal Inhibiti on concentration IGF 1 Insulin like growth factor 1 I B IRS 1 Insulin receptor 1 kDa Kilodalton kg Kilogram kPa Kilopascal mg Milligram mm Millimeter mRNA Messenger ribonucleic acid mTORC1 Mammalian target of rapamycin complex 1 MuRF1 M uscle RING finger protein 1 MV Mechanical ventilation
9 NF B Nuclear factor B nM Nanomolar PBS Phosphate buffered saline PCO 2 Partial pressure of carbon dioxide PCR Polymerase chain reaction PEEP Positive end expiratory pressure PI3K Phosphoinositide 3 ki nase PO 2 Partial pressure of oxygen SEM Standard error margin TUNEL T erminal deoxynucleotidyl transferase nick end labeling Microgram Microliter Micrometer UPP Ubiquitin proteasome pathway VIDD Ventilator induced diaphragm dysfunction
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CALPAIN REGULATES AKT AND NF THE DIAPHRAGM DURING PROLONGED MECHANI CAL VENTILATION By William Bradley Nelson May 2012 Chair: Scott K. Powers Major: Health and Human Performance Mechanical ventilation (MV) is a life saving intervention in patients with acute respiratory failure. However, prolonged MV is associated with numerous clinical indicate that ventilator induced diaphragm dysfunction (VIDD), due to both fiber atrophy and contractile dysfunction, is a key contributor to weaning di fficulties. Previous work has shown that all four major proteolytic systems (calpain, caspase 3, ubiquitin proteasome pathway and autophagy) are activated in the diaphragm during prolonged MV. Interestingly, calpain activation is a required step in VIDD an d experiments done ex vivo provide evidence that calpain may act as a signaling molecule. Our pilot experiments suggest that calpain does indeed act as a unique signaling molecule in a variety of catabolic pathways. Therefore, these experiments tested the hypothesis that calpain plays an important regulatory role in the diaphragm d uring prolonged MV by activating signaling pathways of both Akt and NF established animal model of MV using a specific calpain inhibitor to prevent MV induced activation of calpain in the diaphragm. Indices of calpain activity, Akt and NF signaling were measured in the diaphragm follo wing the experimental protocol. Our
11 study reveals that calpain inhibition preserves Akt phosphorylation and prevents Akt/FoxO3a mediated proteolytic signaling in the diaphragm during MV. Furthermore, tion and NF B activation in the diaphragm following MV. Collectively, these results indicate that calpain activity regulates the Akt/FoxO3a and NF prolonged MV in the diaphragm.
12 CHAPTER 1 INTRODUCTION Mechanical ventilation (MV) is used clinically to maintain blood gas homeostasis for patients unable to maintain adequate alveolar ventilation. It is estimated that nearly 33% of all adults admitted to an in tensive care unit require MV (1) While MV c an be a life saving intervention, prolonged MV can result in difficulties in weaning from the ventilator. Failure to wean patients from MV is a major clinical concern because increased time spent weaning from the ventilator increases the risk of ventilator induced complications. Furthermore, weaning is expensive, weaning procedures account for 40 50% of time spent on the ventilator and it is estimated that almost 30% of mechanically ventilated patie nts undergo weaning difficulty (2, 3) This additional ventilator time results in higher costs to patients, insurance companies, and hospitals. Abundant evidence indicates that prolonged MV promotes diaphragm weakness due to contractile dysfunction and atrophy. This is sign ificant because respiratory weakness is thought to be a primary factor in difficult weaning (4) The negative impact of prolonged MV on the diaphragm has been termed ventilator induced diaphragm dysfunction (VIDD) and it occurs rapidly in patients undergoing MV. Indeed, VIDD has been reported to occ ur in as few as 12 hours in animals and 18 hours in humans (5 7) In this regard, VIDD results from increased protein degradation and decreased protein synthesis during MV (8 10) With respect to protein breakdown, all four major skeletal muscle proteolytic systems, calpain, caspase 3, ubiquitin proteasome pathway and autop hagy lysosomal pathway, have been implicated in VIDD (7, 9, 11) Clearly, understanding th e factors that regulate protein degradation and protein synthesis during MV is essential to develop a therapeutic strategy to prevent VIDD.
13 Calpain, a calcium activated cysteine protease, has been shown to play an essential role in VIDD (8, 12) A lthough all four major proteolytic systems are active in the diaphragm during MV, inhibition of calpain can fully protect the diaphragm from VIDD (8, 12) These resul ts suggest that calpain plays a critical signaling role in VIDD and recent experiments indicate that calpain regulates at least one other proteolytic system in the diaphragm during prolonged MV. Specifically, caspase 3 activation appears to be r egulated by calpain activity in the diaphragm during MV (12) Clearly, in VIDD is unknown. Based on both theory and in vitro experiments, we predict that active calpain can depress protein synth esis signaling and signal an increase in the activation of the other major proteolytic pathways. The theory behind this assertion follows. Previously published in vitro experiments suggest that active calpain can downregulate Akt phosphorylation (13) This is significant because active Akt (i.e., phosphorylated) can depress the activation of both the ubiquitin proteasome pathway and the autophagy lysosomal pathway. Further, active Akt promotes protein synthesis wher eas inactive Akt results in a depression of protein synthesis. More sp ecifically, when phosphorylated, Akt phosphorylates mTOR C1 which initiates a signaling cascade th at promotes protein synthesis (14 16) Furtherm ore, phosphorylated Akt by virtue of its kinase activity, also regulates the activity of a key proteolytic transcriptional act ivator, FoxO3a, through phosphorylation. When phosphorylated, FoxO3 remains in the cytosol in an inactive state. However, unphosp horylated FoxO3 (i.e., activated) translocates to the nucleus to initiate gene transcription necessary for the expression of important
14 proteasome and autophagy proteins (17, 18) gulate Akt phosphor ylation status could impact not only protein synthesis, but also prote olysis isolated to Akt regulation. Indeed, it is also feasible that calpain can in fluence another important proteasome transcriptional activator, NF recent report reveals that NF (19) Moreover, the link between active calpain and NF endogenous i nhibitor of NF (20 22) D increases the transcript i on of important atrophy genes that are involved in the proteolytic activity of t he ubiquitin proteasome system (23) From these observations, we designed these experiments to test the hypothesis that during prolonged MV, calpai n is a signaling molecule in the diaphragm that regulates the activity of Akt and NF integrated specific aims: Aim 1: Will determine the role that increased calp ain activation plays in Akt/FoxO3a proteolytic signaling in the diaphragm during prolonged MV. Hypothesis: Calpain inhibition during MV will preserve Akt phosphorylation, preventing FoxO3a mediated proteolytic signaling in the diaphragm. Aim 2: Will establish if MV induced calpain activation is re quired for NF activation in the diaphragm during prolonged MV. Hypothesis: Inhibition of calpain will prevent NF the nucleus in the diaphragm during MV.
15 CHAPTER 2 LITERATURE REVIEW The diaphragm is the primary muscle of inspiration in all mammals and is essential for normal breathing. It follows that diaphragm dysfunction can impair the ability to maintain adequate alveolar ventilation and gas exchange. In this regard, prolonged mechanical ventilation (MV) is associat ed with diaphragm weakness, which is predicted to be a major contributor to the inability to wean patients from the ventilator. This ventilator induced diaphragm weakness that stems from both atrophy and contractile dysfunction has been termed ventilator i nduce d diaphragm dysfunction (VIDD) (24) Understanding the signaling mechanisms responsible for VIDD is the first step toward developing a therapeutic intervention to protect against VIDD and reduce the risk of wea ning problems. The primary purpose of this review will be to discuss inactivity induced signaling events that regulate proteolysis in the diaphragm during prolonged MV. This review will begin with an introduction to VIDD, followed by a discussion of the potential role that calpain plays as a central regulator in ventilator induced diaphragm atrophy. Ventilator Induced Diaphragm Dysfunction is Clinically Significant MV is a life saving intervention used clinically to sustain adequate alveolar ventilation in patients that are unable to do so on their own. A few of t he most common indications for MV include heart failure, cardiovascular surgery, and respiratory failure due to lung disease. It is estimated that around 33% of all adults in the intensive care u nit require MV (1) While MV is a life saving intervention, withdrawal from MV, or (25, 26) Almost 30% of all MV patients will experience weaning problems that will require additi onal time on the ventilator (3) In those patients
16 that do experience weaning difficulties, weaning procedures can account for up to 50% of the t ime spent on the ventilator (2) Regrettably, between 1 5% of patients fail to wean and m ay become ventilator dependent (27) The inability to wean patient s from MV is an important clinical problem because increased time spent on the ventilator increase the risks of morbidity and mortality for mechanically ventilated patients (24, 25, 28 32) The precise pathological conditions that contribute to wea ning difficulty are unknown Nonetheless, several critical factors have been determined: reduced ventilatory drive, increased work of breathing, cardiac failure and inspiratory muscle weakness and fatigue (33) Of these conditions, evidence now suggests that ventilator induced inspiratory muscle weakness and fatigue (i.e., VI DD) play a critical role in weaning problems (4, 34 39) Ventilator Induced Diaphragm Dysfunction Occurs Rapidly Several animal models (e.g., rats, pigs, rabbits) have demonstrated that prolonged periods of MV resu lt in diaphragm atrophy (7, 9, 40 43) As little as 12 18 hours of MV initiates a 15 30% decrease in rat diaphragm muscl e fiber cross sectional area (7) Importantly, both f ast and slow muscle fibers of the diaphragm atrophy in response to MV (7, 9, 44) In contrast, limb muscles of mechanically ventilated animals fail to show signs of atrophy after 12 to 18 hours of inactivity (7, 9) MV induced diaphragm atrophy progresses at a much faster rate than locomotor muscles during disuse. The same level of atrophy observed in the diaphragm after 12 hours of MV requires approximately 96 hours o f inacti vity in limb muscle (45) Moreover, MV induced diaphragm atrophy occurs at an even fast er rate than denervatio n induced diaphragm atrophy (46) Importantly, this phenomenon is not isolated to animals as atrophy in both fast and slow fibers has
17 also been reported in human subjects expos ed to 18 72 hours of MV (5) Indeed, MV induced diaphragm atrophy represents a rapid and novel type of skeletal muscle atrophy. Prolonged MV not only leads to atrophy of the diaphragm but also decreased force production by diaphragm fibers. This finding was initially reported in a rat model of MV. Specifically, 48 hours of MV resulted in a large (i.e., 60%) reduction in diaphragm maximal tetanic specific force production (i.e., forc e per cross sectional area) (47) Since this initial report, numerous studies have confirmed these findings in rats, pigs, rabbits and baboons following prolonged MV (43, 48 53) This ventilator induced decrease in diaphragm force generation is ti me dependent. For example, 12 hours of MV resulted in an 18% decrease in specific force production wherea s 24 hours of MV resulted in a 48% decrement in specific tension (6) Mechanical Ventilation Promotes Protein Turnover Inactivity induced muscle atrophy occurs in both locomotor and respiratory muscles. While diaphragm atrophy has been reported in numerous MV studies involving both humans and animals, the swift progression (i.e., 12 hours) presents a unique disus e model to investigate the atrophy process. D isuse skeletal muscle atrophy results from an imbalance in muscle protein synthesis and muscle protein degradation, with protein degradation greatly exceeding protein synthesis (54) During MV, mixed muscle protein synthesis in the diaphragm of rats has been reported to d ecrease by 30% in as little as six hours. Furthermore, t here was a 65% reduction in the rate of myosin he avy chain protein synthesis (10) In conjunction with this decrease in protein synthesis, there are reports of increased protein degradation. For example, in vitro me asurements of mechanically ventilated rat diaphragm tissue reveal a 46% increase in diaphragm
18 proteolysis (7, 49) Hence, the imbalance in synthesis and degradation is the primary contributor to MV induc ed diaphragm atrophy. The ability to maintain normal protein synthesis and prevent abnormal protein degradation during MV represents targets whereby therapeutic strategies may be employed. Mechanical Ventilation Activates Proteolytic Pathways Several key proteases co ntribute to skeletal muscle atrophy, specifically, the ubiquitin proteasome pathway (UPP), the autophagy lysosomal pathway (ALP), calcium ac tivated calpain and caspase 3. While e ach system appears to play a distinct role in the proteolytic events during at rophy, all four pathways have been implicated in VIDD. A brief overview of each system and its contribution to VIDD follows. Ubiquitin Proteasome Pathway (UPP) Mediated Proteolysis Traditionally, the UPP has been characterized as the primary proteolytic s ystem responsible for digesting damaged an d misfolded proteins (55) The total proteasome (26S) is mad e of a core subunit (20S) with regulatory subunit s (19S) attached to each end of the 20S core (56 58) The 26S proteasome degrades polyubiquinated proteins that have undergone a repeated three step ubiquination process involving E3 ubiquitin ligases (e.g., MuRF1, atrogin 1). E3 ligases are primarily responsibl e for ubiquinating damaged proteins as a method to target them for degradation. Once ubiquinated proteins are recognized and boun d by the 19S regulatory subunit. T he ubiquinated protein is then moved inside the 20S core in an ATP dependent process. Once i nside the 20S, the targeted protein is then degraded (57, 59) Interestingly, the UPP does not appear to be the initial proteolytic event involved in disassembling proteins from the sarcomere. Actomyosin complexes and other myofilaments must first be cleaved free from the sarcomere, unfolded and ubiquinated
19 for UPP degradation (60 65) This dictates that the potential rate limiting step in muscle protein degradation is the r elease of myofilaments from the sarcomere. In this regard, evidence indicates that both calpain and caspase 3 can promote actomyosin disassociation (60, 64, 66) It follows that activation of these proteases is esse ntial for proteolytic activity that leads to skeletal muscle atrophy. Despite evidence that the UPP cannot cleave intact actomyosin complexes, the UPP does contribute to VIDD. Data from our lab indicates that UPP activity is increased in the diaphragm dur ing MV (67) Nonetheless, work from our lab also reveals that selective pharmacological inhibition of the UPP only partially prevents diaphragm atrophy and contractile dysfunction following 12 hours of MV (unpublish ed). Therefore, other proteolytic systems must also play a role in VIDD. Caspase 3 Mediated Proteolysis Caspase 3 is a member of the caspase family of cysteine a spartate specific proteases (68) Caspases exist as pro caspases and are converted to an active protease following the cleavage of an aspartic acid residue (68) Caspase 3, widely known as a key apoptotic player in numerous cell types, is primarily regulated by three interconnected pathways: the mitochondrial mediated pathway (i.e., caspase 9) the calcium endoplasmic reticulum stress mediated pathway (i.e., caspase 12) and the extracellular death ligand pathway (i.e., caspase 8) (69, 70) Upon activation, the respective upstream caspase can cleave pro caspase 3 into its active form, caspase 3. Once activated, caspase 3 provides the final signal for a cell to undergo DNA fragmentati on and eve ntually apoptosis (71) In addition to its role as an apoptotic trigger, caspase 3 activation promotes protei n degradation and muscle atrophy (9, 66, 72) As previously mentioned, caspase 3 is capable of cleaving intact actomyosin
20 complexes and releasing them from the sarcomere (60, 64, 66) It is also established that oxidatively modified actin and myosin proteins are more susceptib le to caspase 3 proteolysis (72) This finding is particularly important in light of the evidence that reactive oxygen species are required for the activation of capase 3 in the diaphragm during MV (73) Furthermore, caspase 3 has been shown to be essential for myonuclear apop tosis, atrophy and contractile dysfunction in the diap hragm following 12 hours of MV (9) Moreover, MV induced caspase 3 activation in the diaphragm appears to be downstream of both the caspase 9 and caspase 12 path ways, rathe r than the capase 8 pathway (12) Emerging evidence indicates that active calpain participates in the regulation of caspase 3 activation during MV. Briefly, selective calpain inhibition prevented the MV induced activation of caspase 3 and further investigation revealed th at calpain a ctivity appears to regulate two upstream caspase 3 a ctivators, caspase 9 and caspase 12 (12) Calpain Mediated Proteolysis Calpain is a cysteine protease that plays an essential role in VIDD. Indeed, inhibition of calpain provides complete protection against MV induced atrophy in all three myofiber types (i.e., type I, type IIa and type IIx/IIb) and preserves specific force generation in diaphragm (8, 12) signal has been ch aracterized in non muscle cell types (e.g., neurons and retina cells). A more detailed discussion of calpain and its potential signaling role will be presented in a subsequent section. Autophagy Lysosomal Pathway (ALP) Mediated Proteolysis The ALP plays an important role in cellular growth and development, organ elle biogenesis and turnover as well as regulating protein balance in the cell (74)
21 Interestingly, autophagy is a double edged sword: on one side it removes damaged organelles and protein aggregates as a pro survival mechanism; on the other hand, excessive autophagic acti vity can lead to cell death (74) The ALP is made up of membrane bound vesicles, called lysosomes, that contain acid hydrolases that include lipases, phosphatases, glycosidases an d proteases (75) The ALP is activated when lysosomes fuse with double membraned vesicles called autophagosomes. Upon fusion, the acid hydrolases then digest the contents of the autophagosome. This process represent s a compartmentalized method to degrade long lived proteins and organelles (76, 77) Induction of the autophagic response begins with the formation of a small isolation membrane ( phagophore). The phagophore becomes the autophagosome through a procedure that recruits necessary proteins called autophagy proteins (Atg proteins). Beclin 1 is part of the phosphoinositide 3 kinase (PI3K) complex and is an important Atg in this process. Beclin 1 mediates the localization o f other Atg proteins to the phagophore to induce formati on of the autophagosome (78, 79) As such, protein levels of Beclin 1 are known to increase in conditions th at are undergoing autophagy (8 0) The formation of the autophagosome also requires the interaction of other key Atg proteins. Specifically, the necessary conjugation of Atg12 to Atg5 requires both Atg7 and Atg10 (81, 82) The Atg12 Atg5 complex then interacts noncovalently with Atg16; this new complex then induces autophagosome membrane elongation by recruiting LC3 after it has been cleaved by Atg4 (78, 83 85) During the elongation process, the autophag osome sur rounds organelles and cytosolic proteins to be sequestered. At this
22 point the mature autophagosome can then fuse with a lysosome, m aking an autolysosome, which then digest s the contents of the autophagosome (78, 84, 85) The lysosomal proteases used by the autophagosome are called cathepsins and there are four major members: L, B, D and H. One member of the cathepsin family, specifically has been shown to be important in skeletal muscle atrophy Cathepsin L mRNA and protein abundance are upregulated throughout several types of atrophy leading to its identification as an atrophy gene (86 88) Once considered to play a small role in muscle atrophy, the ALP has been demonstrated to play a more important role in atrophy than previously thought (89, 90) Evidence from both human and animal diaphragm tissue now show that the ALP is also active during MV. For example, following pr olonged MV in humans, important autophagy genes were significantly increased in the diaphrag m, specifically LC3, BNIP3 and cathepsin L. Furthermore, e lectron microscopy pictures of human diaphragm also showed increased pres ence of autophagic vesicles (11) Calpain is an Important Regulatory Protease Calpain is a calcium activated cysteine protease. The calpain family contains 15 members that are found in man y different types of tissue (91) Calpa calpain) and calpain 2 (m activation in vitro are both heterodimers made of an 80 kDa subunit and a regulatory 30 kDa subunit, these two represent the most studied calpain mem bers of the family (91) Precise detai ls of calpain regulation remain unclear. Nonetheless, it is established that sustained high levels of cytosolic calcium is a require ment for calpain activation (60) It is also clear that calpastatin is a highly sele ctive endogenous inhibitor of calpain and is typically foun d co localized with calpain (92) It follows that high levels of calpastatin
23 inhibit calpain activation whereas diminished levels of calpastatin promotes calpain activation. When calcium levels reach sufficient levels, calpain and calpastatin dissociate and calpain is activated (60) Further more calpain activation can be promo ted by a caspase 3 mediated de gradation of calpastatin (93 95) A recent VIDD study revealed that pharmacological inhibition of caspase 3 can prevent MV indu ced calpastatin degradation in the diaphragm (12) In vitro calpain has been shown to degrade several cytoskeletal proteins that connect with contractile proteins as well as contractile proteins themselves (64, 96, 97) It has been estimated that every Z disk in a myofiber would be degraded in un der five minutes if all the calpain was to b e activated (60) Based on these reports, calpain has been characterized as a protease that primarily contributes to cytoskeletal protein modifications required for muscle plasticity. Indeed, calpain has been shown to play a prote olytic role in skeletal muscle atrophy as well as exercise adaptations (98 101) Most notably, overexpression of the endogenous calpain inhibitor, calpastatin, reduced loss of muscle mass in mice by 30% after 10 day s o f hindlimb muscle unloading (64) As previously mentioned, calpain is also implic ated in MV induced diaphragm atrophy. Indeed, pharmacological inhibition of calpain prevents MV induced proteolysis, atrophy and contractile dysfunction in the diaphragm muscle (8, 12) These findings support the idea that calpain plays an important role in the muscle remodeling that occurs during prolonged periods of inactivity. Emerging work in non muscle cells suggests that calpain may be an importa nt regulatory protease as numerous substrate proteins are modulated by calpain driven hydrolysis (102) As such, calpain participates as a cellular signal, mediating necessary
24 cell functions such as signal transduction, apoptosis, cell proliferation, cell differentiation as well as membrane fusion and platelet activation (103 107) At the same time, unregulated calpain activity has been implicated in several pathologies, including proper calpain regulation is important to maintaining the overa ll health of the tissue. Calpain as a Signaling Molecule in Skeletal Muscle Growing evidence suggest that calpain can participate in numerous cellular functions in non skeletal mus cle. Interestingly, there is one report that first uncovered a possible signaling role for calpain in skeletal muscle. The experiments revealed a link between calpain and Akt in an ex vivo diaphragm model. When diaphragm tissue was incubated with calcium, phosphorylated Akt (i.e., activated) protein levels decreased significantly below control (13) However, when the diaphragm was exposed to calcium in the presence of a calpain inhibitor, phosphorylated Akt levels re mained elevated, suggesting that calcium activated calpain contributes to Akt inactivation in some manner. A new report done in diaphragm muscle supports the hypothesis that calpain can act as a signal in skeletal muscle in vivo These experiments indicat ed that inhibition of calpain in the diaphragm pr events caspase 3 activation (12) Further more this report also revealed that upstream pro apoptotic pathways (caspase 9, casoase 12 and Bid) were not activated in the diaphragm. These results provide new evidence that calpain acts up stream of apoptotic pathways in diaphragm muscle during MV. Collectively, the Akt and apoptotic pathway data support the novel concept that in skeletal muscle calpain can act as a signal, not simply a remodeling protease. The remaining portion of
25 this rev muscle. Calpain Impacts Akt Signaling Protein kinase B, also known as Akt, holds a central position in modulating skeletal muscle protein synthesis and degradation. Pho sphorylated Akt, the active form of the kinase, can increase protein synthesis by phosphorylating mTOR C1 resulting in the activation of downstream targets that induce protein synthesis (108) Indeed, adult mice tha t conditionally expressed a constitutively active Akt for 14 days doubled their muscle mass (109) Conversely, re search has shown that inactive Akt promotes the unphosphorylation and thus, activation of the transcri ptional activator, FoxO3a (110) Specifically, unphosphorylated FoxO3a is free to translocate to the nucleus and increase transcription of not only important ubiquitin ligases (e.g., atrogin 1) but also autophagy re lated genes (e.g., LC3 and BNIP3) (17, 110) It is through this pathway that we hypothesize calpain can influence protein degradation (Figure 3 1). The mechanism by which calpain can promote dephosphorylation of t he Akt signal is unknown. However, evidence for three possibilities exists. First, one of the known proteolytic substrates of calpain is the insulin re ceptor substrate 1 (IRS 1) (111) This protein is phosphorylated by the insulin like growth factor 1 (IGF 1) receptor at the sarcolemma and leads to activation of phosphoinositide 3 kinase (PI3K) which then activates Akt (112) by phosphorylating it. Degradation of IRS 1 would e ssentially disconnect the IGF 1/Akt signaling pathway. A second possibility is that calpain can dering the kinase inactive (113) Similar to IRS 1 degradation, cleavage of PI3K would disconnect the PI3K/Akt cascade, preventing Akt phosphorylation (i.e., activation). A third possibili ty is
26 that calpain can activate the phosphatase calcineurin through cleava ge of a regulatory subunit (114) Akt is a known target of calcineurin and as such, activated calcineurin can l ead to dephosphorylati on and inactivation of Akt (115) Calpain Can Activate NF Nuclear factor (NF) regulatory expression of many early response genes related to inflammation, infection and tissue injury (20) Furthermore, activated NF to play an important role in disuse muscle atrophy (116) NF NF Rel) transl ocate to t he nucleus. This occurs due to phosphorylation, by the UPP. O therwise NF cytosol inactive t step in activating NF which, upon activation can increase the expression of numerous atrophy causing genes, most notably MuRF1, which leads to increased activity of the UPP (23, 117) and p53 a known pro apopt otic factor (118) In theory, calpain can regulate NF This represents an alternative pathway for degradation and NF mentioned above, it is generally held that the UPP degrades However, in vitro work has also shown that calpain can in fact, to activation of NF (20 22) Therefore, it is feasible that calpain activation can also regulate protein degradation via NF 1) Summary MV is a life saving intervention. However, prolonged periods of MV contribute to difficulty in weaning from the ventilator. It is believed that VIDD, characterized by
27 atrophy and contractile dysfunction, is a primary contributor to weaning difficu lty. While not all of the mechanisms are known behind VIDD, it is known that protein synthesis and degradation in the diaphragm are altered. Furthermore, it is also known that calpain ole has not been determined. There are several possible links between calpain and the decrease in protein synthesis (i.e., Akt) and increased protein degradation (i.e., FoxO3a and NF Clearly, additional experiments that clarify the signaling ro le of c alpain in the diaphragm d uring prolonged MV are important and could provide important insights into possible therapeutic targets to protect against VIDD.
28 Figure 3 1. Proposed calpain signaling pathway in the diaphragm during prolonged mechanical venti lation. Increased calpain activity results in the dephosphorylation of Akt and subsequent activation of FoxO3. FoxO3 activity increases protein degradation via autophagy and ubiquitin proteasome pathway (UPP) through upregulated atrophy genes. Furthermore, we propose and upregulate atrophy genes, leading to increased UPP activity.
29 CHAPTER 3 METHODS Experimental Design Young adult female Sprague Dawley rats were used in these ex periments. Animals were assigned to one o f three experimental groups (eight rats per group): control, 12 hours of MV or 12 hours of MV with a specific calpain inhibitor. The Institutional Animal Care and Use Committee of the University of Florida approved these experiments Control Animals Control animals were acutely anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight). After reaching a surgical plane of anesthesia, the diaphragm was quickly removed and the costal diaphragm was divided into several segments. A strip of the medial costal diaphragm was stored for histological measurements and the remaining portions of the costal diaphragm were rapidly frozen in liquid nitrogen and stored at 80C for subsequent bioche mical analyses Mechanically Ventilated Animals All surgical procedures were perfo rmed using aseptic techniques. Animals in the MV groups were anesthetized with an intraperitoneal injection of sodium pento barbital (60 mg/kg body weight). Animals were then t racheostomized, and mechanically ventilated with a pressure controlled ventilator (S ervo Ventilator 300, Siemens AG; Munich, Germany) for 12 hours with the following settings : upper airway pressure limit: 2 0 cm H 2 O; respiratory rate: 80 breathes per minute ; PEEP: 1 cm H 2 O. The carotid artery was cannulated to permit the continuous measurement of blood pressure and the collection of blood during the proto col. Arterial blood samples (15 0 l
30 per sample) were removed periodic ally and analyzed for arterial P O 2 P CO 2 and pH using an electronic blood gas analyzer (GEM Premier 3 000; Instrumentation Laboratory; Lexington, MA, USA). Ventilator adj ustments were made if arterial P CO 2 exceeded 40 mm Hg. Moreover, arterial P O 2 was maintained > 60 mm Hg throughout the expe riment by increasing the F I O 2 (22 27%). A venous catheter was inserted into the jugular vein for continuous inf usion of sodium pentobarbital ( 10 mg/kg of body weight /hr). Body temperature was maintained at 37C by use of a recirculating water heating blan ket and heart rate was monitored via a lead II electrocardiograph. Continuous care during the MV protocol included lubricating the eyes, expressing the bladder, removing airway mucus, rotating the animal, and passively moving the limbs. Animals also receiv ed an intramuscular injection of glycopyrrolate (0.04 mg/kg of body weight) every two hours during M V to reduce airway secretions. Upon completion of MV, the diaphragm was quickly removed and a section was stored for histochemical analyses, the remaining p ortion was frozen in liquid nitrogen and stored at 80C for subsequent biochemical analyses. Calpain Inhibition To prevent MV induced diaphragm calpain activation, we administered 3 mg/kg body weight of a highly selective calpain inhibitor, SJA 6017 (Calp ain Inhibitor VI, calpain 1 IC 50 : 7.5 nM and calpain 2 IC 50 : 78 nM; N (4 fluorophenylsulfonyl) L valyl L leucinal, EMD Chemicals; Gibbstown, NJ) Previous work from our lab has shown this to be an effective in vivo dose (12) The inhibitor was dissolved in 88% propylene, 10% ethyl a lcohol, 2% benzyl alcohol and given intravenously as a bolus at the begin n ing of MV. Intravenous administration of SJA 6017 has been shown to have a terminal plasma half life of 42 minutes (119)
31 Western Blot Analys is Protein abundance of individual proteins was determined in diaphragm samp les via Western Blot analysis. Briefly, diaphragm tissue samples were homogenized 1:10 (wt/vol) in 5 mM Tris (pH 7.5) and 5 mM EDTA (pH 8.0) with a protease inhibi tor cocktail (Sig ma Aldrich; St. Louis, MO) and centrifug ed at 1500 g for 10 min at 4C. After collection of the resu lting supernatant, diaphragm protein content was assessed by the metho d of Bradford (Sigma Aldrich; St. Louis, MO). Proteins from the supernatant fraction w ere separated via polyacrylamide gel electrophoresis via 4 20% gradient polyacrylamide gels containing 0.1% sodium dodecyl sulfate for 23 min utes at 300 volts. After electrophoresis, the proteins were transferred to nitrocellulose membranes. Nonspecific si tes were blocked for two hours at room temperature in a phosphate buffered saline solution containing 0.05% Tween and 5% nonfat milk. Membranes were then incubated overnight at 4C with primary antibodies directed against the protein of interest. Specifica lly, we assessed calpain activit y by measuring protein abundance of active calpain II spectrin 145 kDa calpain specific cleavage product. We measured Akt activity by measuring the total and phosphorylated (Ser 473) proteins of Akt We also measured Akt signaling by assessing MuRF1 and atrogin 1 proteins. NF After washing, a chemiluminescent system was used to detect labeled proteins (GE Healthcare ; Piscataway, NJ ). Membranes were developed using autoradiography fi lm and images of the film were captured and analyzed using the 44 0CF Kodak Imaging System (Kodak; New Haven, CT). To control for protein loading and transfer differences, membran es were stained with Ponceau S. Ponceau S stained membranes were scanned and t he
32 lanes were quantified (440CF imaging system, Kodak, New Haven, CT) to normalize Western blots to protein loading. RNA I solation and cDNA synthesis Total RNA was isolated from muscle tissue with TR Izol Reagent (Life Technologies; Carlsbad, CA) according (g/mg muscle weight ) was evaluated by spectrophotometry. RNA (5 g) was then reverse transcribed with the Superscript III Firs t Strand Synthesis System for reverse transcription PCR (Life Technologies), usin g oligo(dT)20 primers and the protocol outlined by the manufacturer. Real Time Polymerase Chain R eaction One l of cDNA w as added to a 25 l PCR for real time PCR using Taqman chemistry and the ABI Prism 7000 Sequence Detection system (ABI; Foster City, C A). Relative quantification of gene expression was performed using the comparative Glucuronidase, a lysosomal glycoside hydrolase, was chosen as the reference gene based on previous work showing unchang ed expression with our experi mental manipulations (126). BNIP 3, LC3, MuRF1, atrogin 1, and FoxO3a mRNA transcripts were assayed using predesigned rat primer and probe sequences commercially available from Applied Biosystems (Assays on Demand). TUNEL Analys is for Apoptosis Myonuclear apoptosis was determined by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) using a histochemical fluorescent detectio n kit (Roche Applied Scientific; Shandon Cryotome cryost at (Life Sciences International; England) from embedded
33 diaphragm tissue. Sections were fixed usi ng in a 4% formaldehyde solution, washed, and permeabi lized with 0.1% T riton X 100 in 0.1% sodium ci t rate solution. For identification of cell membranes, tissues were incubated with a rabbit anti dy strophin antibody (Thermo Scientific; Freemont, CA ), and secondary conjugated to Rh odamine Red (Invitrogen; Eugene, OR ). Tissue s ections were then incubated with the TUNEL enzyme label solution and sealed with a Vectashield DAPI mounting medium for detection of nuclei (Vector Laboratories; Burlingame, CA) TUNEL stained tissue sections were imaged using a Zeiss fluorescent microscop e (Thornwood, NY). Fluorescent images for DAPI (nuclei), Rhodamine (dystrophin), and FITC filters (TUNEL positive) were combined using IP Lab software (Scanalytics, Inc.; Fairfax, VA). TUNEL positive nuclei were counted and normalized to tissue cross sectio nal area. 20S Proteasome Activity Assay A section of the ventral costal diaphragm was homogenized 1:10 (vol : vol) (5 mM Tris HCL, pH 7.5; 5 mM ED TA, pH 8.0) and centrifuged at 1500 g for 10 min utes at 4C. The c ytosolic fraction was centrifuged at 10,000 g for 10 min utes at 4C followed by an additional spin of the supernatant at 100,000 g for 1 h our at 4C The in vitro chymotrypsin like activity of the 20S proteasome was measured fluorometrically using techniqu es described by Stein et al (120) In Vitro Analysis of Calpain Specificity To determine if our chosen inhibitor could successfully inhibit calpain while also not inhibiting the 20S proteasome, we designed an in vitro experiment adapted from Pereira et al (121) Briefly, u sing pur chased purified active 20S proteasome we measured the cl eavage of fluorogenic sub strate when exposed to concentrations of inhibitor similar to our calculated in vivo concentrations, based on 70% body water.
34 Sample fluorescence, an indicator of substrate cleavage, was measured after 20 minutes of incubating substrate, active enzyme, and the inhibitor. Trolox Equivalency Antioxidant Capacity Assay SJA 6017 (2 mg/ml) was dissolved in 88% propylene glycol, 10% benzyl alcohol 6017 was mixed with 2 m l of ABTS ( 2,2' azino bis(3 ethylbenzothiazoline 6 sulphonic acid ) buffer solution (50 mM glycine, 10 6017 of 0.1 mg/ml and incubated for 2 minutes. The sample was then measured with a spectrophotometer at a wavelength of 414 n M and compared to a Trolox based standard curve. Statistical Analysis Comparisons between groups for each dependent variable were made by a one way analysis of variance (ANOVA) and w hen appropriate, a Tukey honestly significant difference test was perform ed post hoc. Significance was established at p < 0.05. D ata are presented as means standard error margin (SEM)
35 CHAPTER 4 RESULTS Physiological Responses to P rolonged MV To ensure that our mechanical ventilation (MV) protocol was successful in mainta ining homeostasis, we measured arte rial blood pressures, arterial PCO 2 arterial P O 2 and arterial pH in all animals at the beginning of the experiments an d at various times during MV. Our results (mean SEM) confirm that arterial blood pressure (95.8 2. 4 mm Hg), arterial P O 2 (83.3 3.3 T o rr), arterial P CO 2 (37.1 0.9 T orr) and pH ( 7.42 0.01) were maintained during MV. Furthermore, during the course of MV, no significant (p<0.05) changes occurred in the body weights of our animals. Pharmacological In hibition of Calpain Activity in the Diaphragm We investigated the role of calpain signaling in the diaphragm during MV by using a highly selective pharmacological inhibitor of calpain activity (SJA 6017) Calpain activity in the diaphragm was assayed usin g two distinct but complementary markers of active calpain: 1) determination of the protein abundance of the active form of calpain and 2) measurement of the calpain II spectrin. In the presence of sufficient concentrations o f intracellular calcium, calpain undergoes autolysis resulting in an active form of calpain 1 which can be detected via western blotting (60) Therefore, t he appearance of the active calpain 1 band indicates calpain activity at the time of sampling. Importantly, o ur data in dicate that 12 hours of MV resulted in a significant increase in the presence of active calpain 1 in the diaphragm ( Figure 4 1). Additionally, we assessed the activity of calpain throughout the 12 hour experimental protocol by measuring the calpain specifi c 145 kDa degradation product of II spectrin. This calpain specific protein breakdown product accumulates as a result of
36 calpain activity and has a long half life (i.e., 24 hours) (122) As such, this represents an excellent biomarker of calpain activity during the hours prior to sampling. Importantly, our experiments reveal a sign ificant increase in the calpain specific degradation product II spectrin in the diaphragm following 12 hours of MV ( Figure 4 2). We used SJA 6017 to s electively inhibit calpain activity in order to evaluate results indicate that treatment of animals with SJA 6017 prevented the MV induced increases in calpain activity in the diaphragm ( Figure s 4 1 and 4 2) Calpain Inhibition Maintains Akt P hosphorylation Traditionally, calpain is viewed as a protease responsible for degrading cytoskeletal proteins in skeletal muscle. To determine if active calpain can also participate in re gulatory signaling in skeletal muscle, we measured the protein abundance of phosphorylated Akt (Serine 473) in the diaphragm after 12 hours of MV. Our data reveal that 12 hours of MV resulted in a significant decrease in phosphorylation of Akt in the diaph ragm ( Figure 4 3) with no changes in total Akt protein abundance (Figure 4 4) W hen animals were treated with the calpain inhibitor during MV, Akt phosphorylation in the diaphragm was preserved at levels equal to control. These results suggest that active calpain p revents Akt phosphorylation in the diaphragm during prolonged MV Calpain Inhibition Prevents Akt/FoxO3a Atrophy S ignaling Because inactive (unphosphorylated) Akt can promote protein degradation through FoxO3a signaling, we next determined if Fo xO3a gene transcription is promoted by calpain activity. Our results show a significant increase in FoxO3a mRNA expression in the diaphragm following 12 hours of MV. Importantly, our findings also
37 reveal that inhibition of calpain prevents the MV induced i ncrease in FoxO3a mRNA expression in the diaphragm ( Figure 4 5). Since FoxO3a is a transcriptional activator that regulates the expression of essential atrophy genes we determined if active calpain promotes an increase in transcription of FoxO3a target ge nes. Our findings reveal that the mRNA expression of both MuRF1 and atrogin 1, important muscle specific E3 ligases and FoxO3a target genes, were significantly increased in the diaphragm following 12 hours of MV (F igur e 4 6 and Figure 4 7 ). We also measure d the mRNA expression of three other important proteolytic proteins that are target genes of FoxO3a, specifically BNIP3, cathepsin L and LC3 Our results indicate that 12 hours of MV resulted in a sig nificant increase in mRNA of all three of these proteins in the diaphragm Moreover, inhibition of calpain activity prevented t he MV induced increases ( Figures 4 8 through 4 10). To determine if these mRNA changes reflected changes in protein expression, w e also measured the protein abundance of MuRF1 and a tr ogin 1 in the diaphrag m following 12 hours of MV. Our data re veal a significant increase in both of these E3 ligases. In contrast when animals were treated with the calpain inhibitor during MV, there was no increase in protein abundance of MuRF1 or atrogi n 1 in the diaphragm (Figure 4 11 and Figure 4 12 ). Collectivel y, these findings provide evidence that active calpain increase s the activity of FoxO3a transcription in diaphragm during MV. Calpain Inhibition Prevents Apoptosis Given that active calpain has been shown to promote apoptosis in cardiac myocytes, we determined whether active calpain was a requirement for MV induced myonuclear apoptosis in the diaphragm. Using a TUNEL assay, a method that labels apoptotic DNA strand breaks, we report a significan t increase in myonuclear apoptosis
38 as indicated by increased TUNEL positive myonuclei in the diaphragm. However, pharmacological inhibition of calpain prevented this MV induced increase myonuclear apoptosis in the diaphragm (Figure 4 13). These findings de monstrate that active calpain participates in pro apoptotic signaling in the diaphragm during prolonged MV. Active Calpain Promotes 20S Proteasome A ctivity Since active calpain can regulate both the mRNA expression and protein abundance of the important E 3 ligases, MuRF1 and atrogin 1, we sought to establish whether active calpain impacts the proteolytic activity of the 20S proteasome. In agreement with previous work, we report that 12 hours of MV resulted in a significant increase in 20S proteasome activi ty in the diaphragm (7, 49) N ever theless, inhibition of calpain activity in the diaphragm prevented this MV induced increase in 20S proteasome activi ty in diaphragm (Figure 4 14). Although the mechanism remains unc lear, these results suggest that active calpain augments the proteolytic activity of the 20S proteasome Evidence that SJA 6017 is a Selective Calpain Inhibitor St udies using pharmacological inhibitors are often criticized due to concerns about off target effects In this regard, previous work from our lab has shown that the calpain inhibitor used in these experiments (SJA 6017) does not inhibit caspase 3 activity in vitro (12) In the current experiments, we performed an in vitro experiment using purified active 20S proteasome to determine if SJA 6017 directly inhibits 20S proteasome activity. Importantly, our data demonstrate that SJA 6017 does not inhibit 20S proteasome substrate cleavage (Figure 4 15) These results demonstrate that our findings indicating that inhibition of cal pain is associated with a down regulation of the 20S proteasome are not due to off target of the calpain inhibitor.
39 Calp egradation In vitro work has revealed that calpain can degrade endogenous inhibitor of NF (20) Degradation of is an important step in NF because it leads to increased NF proteolysis through activation of atrophy genes. To investigate whe ther active calpain can regulate NF via degradation we measured the in diaphragm following 12 hours of MV. Our results show that MV induces a significan t 16 ). However calpain inhibition prevented this MV induced decrease in equal to control. Active Calpain Regulates NF B Activity Because I is upstream of NF calpain mediated I deg radation leads to increases in NF activity. As a marker of NF activity, we measured DNA binding of the NF family member p50. We report that 12 hours of MV resulted in significant increase in p50 DNA binding in the diaphragm. Conversely, inhibition of active calpain prevented the MV induced increase in p50 DNA binding (Figure 4 17). We interpret these findings as evidence that active calpain is required for MV induced NF activation in the diaphragm. SJA 6017 Does Not Exhibit Antioxidant Capacity Previous work from our lab has demonstrated that oxidative stress is a requirement for calpain activation and VIDD (49, 73) To determine if the calpain inhibitor used in these experiments acted as an antioxidant, w e performed a Trolox equivalency antioxidant capacity assay. Our results demonstrate that our chosen inhibitor, SJA 6017, did not quench the oxidant solution used in the assay, suggesting
40 that SJA 6017 does not possess antioxidant capability. These results support earlier in vivo findings from our lab indicating that SJA 6017 does not function as an antioxidant by preventing oxidative damage in the diaphragm during MV (12)
41 Figure 4 1. Protein level of the active form of calpain 1 in diaphragm of experimental groups Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups
42 Figure 4 II spectrin breakdown product (SBPD) in II II spectrin. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups.
43 Figure 4 3. Protein lev els of phosphorylated Akt in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups.
44 Figure 4 4. Pr otein levels of total Akt in diaphragm of experimental groups. Values are m eans SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor.
45 Figure 4 5. mRNA levels of FoxO3 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhib itor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups.
46 Figure 4 6 mRNA levels of MuRF1 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups
47 Figure 4 7. mRNA levels of a trogin 1 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = di fferent (p<0.05) from both Control and MV+Calpai n Inhibitor groups
48 Figure 4 8 mRNA levels of BNIP3 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups.
49 Figure 4 9 mRNA leve ls of c athepsin L in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. 0.05) from MV group
50 Figure 4 10 mRNA levels of LC3 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibi tor groups.
51 Figure 4 11 Protein levels of MuRF1 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups.
52 Figure 4 12 Protein levels of atrogin 1 in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups
53 Figure 4 13. TUNEL positive nuclei in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor group s.
54 Figure 4 14 Chymotrypsin like 20S proteasome activity in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = different (p<0.05) from both Control and MV+Calpain Inhibitor groups.
55 Figure 4 15. In vitro 20S proteasome activity. Bars represent end point fluoresce nce after 20 minute incubation. Sub, substrate; 20S, 20S proteasome; Calp Inhib, calpain inhibitor. Groups with different letters are statistically diff erent from each other, (p<0.05).
5 6 Figure 4 16 means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = diff erent (p<0.05) from both Control and MV+Calpain Inhibitor groups.
57 Figure 4 17. p50 DNA binding activity in diaphragm of experimental groups. Values are means SEM. Con, control; MV, mechanical ventilation; Calp, calpain; Inhib, inhibitor. = diff erent (p<0.05) from both Control and MV+ Calpain Inhibitor groups.
58 CHAPTER 5 DISCUSSION Overview of the Principal Findings Prolonged mechanical ventilation promotes the rapid development of VIDD and activation of calpain is required for this rapid onset of VIDD (12) The mechanism(s) to explain why calpain plays a cr itical role in VIDD remains unknown. However, the current data provide novel insights into the functions that calpain performs in VIDD. Specifically, our data indicate that during prolonged MV active calpain regulates both Akt and NF signaling in the di aphragm Indeed our findings support the hypothesis that MV induced calpain activation promotes increased Akt/Fo xO3a signaling in the diaphragm, resulting in increased expression of key proteolytic proteins involved in both the ubiquitin proteasome pathwa y (UPP) and autophagy Furthermore, our results reveal that active calpain can also activate NF Collectively these findings indicate that active calpain plays an important signaling role in the diaphragm during prolonged MV A detailed dis cussion of the findings follows Calpain Acti vity Regulates Akt Phosphorylation The phosphorylation status of Akt is a pivotal factor in both catabolic and anabolic reg ulation in skeletal muscle (123) In anabolic conditions, phosphorylated (active) Akt incre ases protein synthesis via mTORC1 mediated signaling, resulting in elevated protein synthesis Conversely, during catabolism, un phosphorylated (inactive) Akt permits FoxO3a nuclear translocation and DNA binding which upregulates gen e expression of several atrophy related genes involved in both the UPP and autophagy. Therefore the status of Akt phosphorylation is an important metabolic cross r oad in skeletal muscle regulating both anabolism and catabolism
59 Our group has previously shown that 18 hours of MV decreases phosphory lated Akt in the diaphragm and our current results confirm these findings (44) Importantly, the present experiments are the first to demonstrate that in vivo inhibition of calpain preserves Ak t phosphorylation in the diaphragm during prolonged MV Our results confirm previous experimental results using an ex vivo diaphragm model (13) Together, these ex vivo results and our in vivo findings support the c oncept that Akt phosphorylation in skeletal muscle is regulated, at least in part, by calpain activity Currently, t he precise signaling mechanism responsible for calpain influence on Akt phosphorylation remains unknown. Nonetheless, there are at least t hree different possibilities whereby calpain could influence Akt phosphorylation in skeletal muscle First, insulin receptor substrate 1 (IRS 1) is a proteolytic subs trate of calpain (111) IRS 1 is phosphorylated b y the insulin like growth factor 1 (IGF 1) receptor at the sarcolemma and leads to activation of phosphoinositide 3 kinase ( PI3K ), which then activates Akt through phosphorylation (112) Degradation of the IRS 1 pro tein would theoretically disconnect the IGF 1/Akt phosphorylation pathway. A second possibility also relies on calpain targeting an upstream protein of Akt. Recently, an in vitro study concluded that calpain can ren dering the kinase inactive (113) Similar to IRS 1 degradation, cleavage of PI3K would disc onnect the PI3K/Akt cascade, preve nting Akt phosphorylation and activation A third possibility is that calpain can activate the phosphatase calcineurin through cleava ge of a regulatory subunit (114) Akt is a known target of calcineurin and as such, activated calcineurin lead s to dephosphorylati on and inactivation of Akt (115)
60 In summary, our novel findings demonstrate that MV induced activation of calpain in the diaphragm plays an important role in the regulation of Akt phosphorylation status. Indeed, pharmacological inhibition of calpain activation prevented the MV induced dephosphorylation of Akt in the diaphragm. These findings are consistent with the concept that active calpain plays an important role in the regulation of Akt modulated signaling in the diaphragm during prolonged MV. Calpain Activity Regulates Akt/FoxO3a Proteolytic Signaling Our finding that Akt phosphorylation status can b e regulated, at least in part, by balance of cells. When activated through phosphorylation, Akt can initiate protein synthesis by increasing translation. Conversely, when unphosphorylated, Akt promotes protein degradation through FoxO3a mediated signaling. A growing number of studies have established an important role for Akt/FoxO3a signaling in skeletal muscles undergoing atrophy (1 7, 124, 125) As mentioned earlier, un phosphorylated Akt leads to activated FoxO3a which upregulates the expression of critical atrophy genes including ubiquitin ligases and several autophagy genes. Active Calpain Increases the Expression of Ubiquitin L igases Muscle specific E 3 ubiquitin ligases, MuRF1 and a trogin 1, play a critical role in UPP dependent muscle atrophy (126) Ubiquitin ligases target specific proteins for degradation by tagging the prote in with ubiquitin which directs the protein to the proteasome. The regulatory role of E3 ligases in muscle wasting was first reported in denervation studies. These studies revealed that both MuRF1 and a trogin 1 knockout animals were resistant to denervati on induced muscle atrophy (126) Moreover, cell
61 culture experiments have de monstrated that overexpression of either MuRF1 or a trogin 1 is sufficient to induce atrophy (126) Prior work in VIDD has shown that both MuRF1 and atrogin 1 mRNA expression was significantly increase d in the diaphragm following 18 hours of MV (44) Our findings are in agreement with these studie s. Specifically, MuRF1 and atrogin 1 mRNA expression increased significantly in th e diaphragm following 12 hours of MV Importantly our results reveal that both MuRF1 and a trogin 1 gene expression is regulated by active calpain Indeed, inhibition of calp ain activity in the diaphragm prevented the MV induced increases in both MuRF1 and atrogin 1 mRNA levels. Autophagy Gene Expression is Regulated by Active Calpain Autophagy is a tightly regulated lysosomal pathway for degrading cytoplasmic proteins and o rganelles. Basal level s of autophagy are important for normal c ell function, recycling old and damaged proteins. However, excessive autophagic activity can lead to pathological conditions such as increased apoptosis and cellular atrophy. O ur current result s agree with previous findings, in that autoph agy is increased in the human diaphragm following MV (11) However, our experiments also provide new and important information regarding the role that calpain plays in r egulating the expression of key autophagy genes. BNIP3 an apoptotic and autophagy gene, initiates apoptosis by translocating to the mitochondria and contributing to the formation of the mitochondrial pe rmeability transition pore (127) While the mechanism (s) for responsible for activating BNIP3 mediated autophagy is unknown, i t appears that BNIP3 may potentially target apoptotic mitochondria for autophagic degradation. Experimental evidence has shown that overexpr ession of BNIP3 leads to increased removal of mitoch ondria in cardiac
62 myocytes (127) Our results show a significant increase in BNIP3 mRNA in diaphragm following 12 hours of MV Importantly, pharmacological inhibit ion of calpain activity prevented this MV induced increase in BNIP3 mRNA levels in the diaphragm. Moreover, our results reveal an increase in the number of TUNEL positive nuclei in the diaphragms of animals exposed to MV, indicating increased myonuclear ap optosis. Similar to our findings with BNIP3 mRNA expression, our data reveal that MV induced myonuclear apoptosis in the diaphragm was prevented when calpain activity was inhibited. Importantly, these findings agree with prior results indicating that both myonuclear apoptosis and autophagy are increased in the diaphragm during MV (9, 11) Nonetheless, the current experiments provide the first evidence that calpain plays an important role in MV induced BNIP3 mediated apoptosis and autophagy in the diaphragm Autophagy begins with formation of the autophagosome, a process where Beclin 1 mediates the localization of essential autophagy ( Atg ) proteins t o the pre autophagosome (78, 79) Once formed, the autophagosome participates in an elongation proc ess, which requires active LC3 (mammalian homolog of yeast Atg 8) (78, 83, 85, 128) Our results show a significant increase in LC3 mRNA in diap hragm following 12 hours of MV, suggesting that prolonged MV results in increased autophagosome formation I nhibition of calpain activity prevented this increase in LC3 mRNA, indicating that the LC3 mediated step in autophagosome formation is regulated by calpain During the LC3 mediated autophagosome elongation process, the autophagosome sequesters both organelles and cytosolic proteins At this point, the
63 mature autophagosome fuses with a lysosome, forming an autolysosome, which digest s the sequestered contents of the autophagosome (78, 84, 128) This digestion is carried out by essential proteases that reside inside the lysosome. One lysosomal protease in particular, cathepsin L, has been shown to be important in skeletal muscle atrophy. As a FoxO 3a transc riptional target gene, cathepsin L has been shown to participate in disuse skeletal muscle atrophy; both mRNA and protein abundance are increased in response to disuse (86 88, 129) Our r esults are consistent with previous reports, cathepsin L mRNA was significantly increased in the diaphragm during MV. Furthermore, our data suggest that cathepsin L transcription in the diaphragm is regulated by calpain activity as inhibit ion of calpain prevented the MV induced increase in diaphragm expression of cathepsin L mRNA MV Induced Calpain Activation Regulates NF Activity NF plays an important role in muscle atrophy. Indeed, NF activation is both sufficient and required for skeletal muscle atrophy in cachexia and disuse experimental models (23, 117, 130 132) NF is a family of five transcription factors that, upon activation, form dimers, which translocate to the nucleus and in crease transcription of important atrophy and apoptotic genes (133 136) Given the important role of NF NF New evid ence indicates that NF (19) Furthermore, it appears that MV induced oxidative stress is required for NF activation in diaphragm during MV (19) Although the data from these experiments does not establish a mechanistic link between oxidative stress and NF activation, the authors theorized that oxidative stress mediated calpain activation could link oxidative
64 stress to NF that calpa in is activated b y oxidants during MV (73) Moreover, evidence from in vitro experiments have determined that calpain is capable of promoting NF deg rading (20 22, 137) Therefore, based on these previous findings, we hypothesized that MV induced calpain activation can activate NF Results f rom our current experiments support this prediction. Following 12 hours of MV, we observed a significant decrease in the abundance of I in the diaphragm, indicative of enhanced I degradation and indirect evidence of increased NF activity. Furthe rmore, inhibition of calpain activity prevented the degradation of I in the diaphragm that is associated with prolonged MV. While our evidence implicates calpain as the protease responsible for degradation, another possibility exists. In skeleta l muscle wasting, a commonly accepted mechanism for kinase (IKK) and ubiquinated, is then degraded by the UPP. Although our data indicate that active calpain is upstream of degradation our data cannot determine whether calpain or the ubiquitin proteasome system is responsible for the degradation. Indeed, our findings demonstrate that inhibition of calpain results in decreased activity of the UPP. Given that our in vitro experimen ts indicate that the calpain inhibitor (SJA 6017) does not inhibit proteasome activity, it appears that active calpain can promote an increase in proteasome activity through increased expression of proteasome proteins by FoxO3a and/or NF signaling; as b oth transcriptional activators have been shown to activate several proteasome genes (138) Regardless of
65 which mechanism degrades it appears that active calpain is upstream of degradation. As mentioned previously, NF is a family of five transcription factors that upon activation dimerize and translocate to the nucleus to initiate gene transcription of atrophy genes. One of those transcription factors, p50, has been shown to be essential for disuse mu scle atrophy (131) Indeed, p50 has been shown to have a significant number (~200) of transcriptional target genes in disuse muscle atrophy (131) Therefore, nuclear localization of p50 is considered to be an excellent biomarker of NF acti vation during skeletal muscle disuse atrophy. To determine if NF activity is regulated by calpain, we measured DNA binding of p50 in diaphragm following 12 hours of MV. These results confirm that NF activity is regulated by calpain. Indeed, inhibitio n of calpain activity prevented the MV induced increase in p50 DNA binding in the diaphragm of MV animals. Together, this evidence suggests that calpain activation is a requirement for MV induced increased NF signaling in the diaphragm. Critique of the Experimental Model Because of the invasive nature of obtaining diaphragm muscle samples from humans, an animal model is required to perform me chanistic experiments to determine the signaling pathways that promote VIDD We selected the rat as the experimen tal animal in these experiments for several reasons. First, the anatomical features and function of the rat diaphragm are both similar to the human diaphragm (139, 140) Second, the fiber type composition of the rat and human diaphragm are comparable (5, 141) Finally, the time course of MV induced atrophy in the rat and human dia phragm are also simi lar (5, 7)
66 The calpain inhibitor utilized in these experiments was chosen due to its ability to effectively inhibit calpain in vivo and because of its high level of specificity. Our in vitro results, both past and present, reveal that using the peak in vivo concentrati ons of the inhibitor, SJA 6017 does not inhibit caspase 3 or the 20S proteasome activity Collectively, these findings confirm the high level of selectivity of our pharmacological inhibitor and demons trate that our results are not due to off target pharmacological effects. We selected sodium pentobarbital as the general anesthetic in these experiments because of evidence that in low doses, it does not negatively impact diaphragm contractile function, does not promote diaphragm atrophy and is not associated with oxidative stress in skeletal muscle (7, 47, 49, 142) We used acutely anesthetized animals as controls in these experiments because our lab has previousl y demonstrated that prolonged exposure to anesthesia does not promote diaphragm contractile dysfunction or atrophy in spontaneously breathing animals (7, 142) Conclusions and Future Directions These experiments pr ovide the first in vivo evidence that c alpain regulates both Akt and NF muscle during MV. Specifically, our results demonstrate that calpain inhibition preserves Akt phosphorylation and prevents Akt/FoxO3a mediated proteolytic signaling in the diaphragm dur ing MV. Furthermore, and increased NF signaling in the diaphragm following MV. Collectively, these are novel and important findings indicating that calpain serves an important signaling role i n the development of VIDD. role in skeletal muscle atrophy was relatively unknown. Historically, in skeletal muscle,
67 calpain had been primarily classified as a protease re sp onsible for degrading large muscle structural proteins (e.g., titin and nebulin) during periods of decreased or increased contractile activity. The current findings greatly expand our knowledge of remodeling Importantly o ur results provide eviden ce that calpain is a signal ing molecule that regulates the activity of both the UPP and autophagy in VIDD. Furthermore, our previous work has already revealed that calpain regulates caspase 3 activity in the diaphragm during MV (12) Taken together, this pri or work and the current evidence indicate that calpain plays a central role in the regulation of three major proteolytic systems during VIDD. Future studies should focus on two areas. First, efforts should be made to understand the mechanisms of how calpa in is activated in the diaphragm during MV. Understanding the mechanis ms of calpain activation is essential to developing safe an d effective countermeasures to prevent central proteolytic signaling role occurs in other types of skeletal muscle wasting (e.g., cancer cachexia and sepsis) Understanding the complex signaling role of calpain activit y in skeletal muscle could provide possible opportunities to develop therapeutic targets against disorders that promote skeletal muscle wasting.
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79 BIOGRAPHICAL SKETCH W. Bradley Nelson, son of Scott and Carrie Nelson, was born in Provo, Utah He grew up in the state of Washington and in 1998 graduated from Governor John. R. Rogers High School in Puyallup, Wash ington. He began his undergraduate education in the fall of 1998 at Brigham Young University in Provo, Utah. After his first semester, he served a two year mission for the Church of Jesus Christ of Latter Day Saints in Puerto Rico. In 2001 he returned to B righam Young University and in April 2004 graduated with a Bachelor of Science degree in physical education, with an exercise science emphasis. Following graduation, Brad remained at Brigham Young University to begin his graduate education with Dr. Gary Ma ck in the department of Exercise Science. His research focused on exercise induced plasma volume expansion as well as cutaneous blood flow regulation. He received his Master of Science degree in 2007 in physical education, with a specialization in exercise physiology. After which, he began a doctoral program at the University of Florida in Gainesville, Florida with Dr. Scott P owers in the department of Applied Physiology and Kinesiology. His research centered on the role of the enzyme calpain in ventilator induced diaphragm atrophy. He graduated in May 2012 with a Doctor of Philosophy degree in exercise physiology.