<%BANNER%>

Overexpression of HO-1 Attenuates MV-Induced Atrophy of the Diaphragm

Permanent Link: http://ufdc.ufl.edu/UFE0021673/00001

Material Information

Title: Overexpression of HO-1 Attenuates MV-Induced Atrophy of the Diaphragm
Physical Description: 1 online resource (71 p.)
Language: english
Creator: Falk, Darin J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mechanical ventilation (MV) is the primary means of support used in patients with respiratory muscle failure. The removal of patients from MV is termed 'weaning' and problems in weaning occur frequently. Importantly, weaning difficulties are attributed to diaphragmatic weakness characterized by atrophy and contractile dysfunction. Oxidative stress contributes to MV-induced diaphragmatic weakness. However, the sequence of events leading to oxidative stress has not been fully elucidated. A key stress sensitive enzyme, heme oxygenase-1 (HO-1), is rapidly induced in the diaphragm during MV. Paradoxically, HO-1 may function either as a pro- or antioxidant and the role that HO-1 plays in MV-induced oxidative stress in the diaphragm is unknown. To address this question we mechanically ventilated rats for 12 or 18 hours (MV) with subsets of animals that combined MV along with a HO-1 inducing agent, hemin (MVH); or MV with a HO-1 activity inhibitor, CrMPIX (MVI). Indices of oxidative stress, proteolytic activation, and atrophy were measured in the diaphragm following the experimental protocol. Our study reveals that hemin-induced elevation of HO-1 during MV provides protection against oxidative injury, proteolytic activation, and diaphragmatic atrophy. Specifically, hemin administration preserves glutathione levels and prevents lipid peroxidation in the diaphragm during MV. Further, overexpression of HO-1 decreased the MV-induced activation of calpain and caspase-3 in the diaphragm. Finally, the hemin-induced resistance to oxidative stress and protease activation in the diaphragm, led to an attenuation of diaphragmatic atrophy following 18 hours of MV. We conclude that HO-1 does not function as a pro-oxidant in the diaphragm but instead provides antioxidant and cytoprotective properties during MV.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Darin J Falk.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021673:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021673/00001

Material Information

Title: Overexpression of HO-1 Attenuates MV-Induced Atrophy of the Diaphragm
Physical Description: 1 online resource (71 p.)
Language: english
Creator: Falk, Darin J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mechanical ventilation (MV) is the primary means of support used in patients with respiratory muscle failure. The removal of patients from MV is termed 'weaning' and problems in weaning occur frequently. Importantly, weaning difficulties are attributed to diaphragmatic weakness characterized by atrophy and contractile dysfunction. Oxidative stress contributes to MV-induced diaphragmatic weakness. However, the sequence of events leading to oxidative stress has not been fully elucidated. A key stress sensitive enzyme, heme oxygenase-1 (HO-1), is rapidly induced in the diaphragm during MV. Paradoxically, HO-1 may function either as a pro- or antioxidant and the role that HO-1 plays in MV-induced oxidative stress in the diaphragm is unknown. To address this question we mechanically ventilated rats for 12 or 18 hours (MV) with subsets of animals that combined MV along with a HO-1 inducing agent, hemin (MVH); or MV with a HO-1 activity inhibitor, CrMPIX (MVI). Indices of oxidative stress, proteolytic activation, and atrophy were measured in the diaphragm following the experimental protocol. Our study reveals that hemin-induced elevation of HO-1 during MV provides protection against oxidative injury, proteolytic activation, and diaphragmatic atrophy. Specifically, hemin administration preserves glutathione levels and prevents lipid peroxidation in the diaphragm during MV. Further, overexpression of HO-1 decreased the MV-induced activation of calpain and caspase-3 in the diaphragm. Finally, the hemin-induced resistance to oxidative stress and protease activation in the diaphragm, led to an attenuation of diaphragmatic atrophy following 18 hours of MV. We conclude that HO-1 does not function as a pro-oxidant in the diaphragm but instead provides antioxidant and cytoprotective properties during MV.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Darin J Falk.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021673:00001


This item has the following downloads:


Full Text

PAGE 1

OVEREXPRESSION OF HO-1 ATTENUATES MV-INDUCED ATROPHY OF THE DIAPHRAGM By DARIN JAMES FALK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

PAGE 2

2007 Darin James Falk 2

PAGE 3

To the numerous teachers and professors who co ntributed to my education and to my wife, friends and family for their continuous support 3

PAGE 4

ACKNOWLEDGMENTS First and foremost, I thank my mentor Dr. Scott Powers for his continuous support and guidance during my graduate studies. Watchi ng the commitment and enthusiasm that Dr. Powers brings to work each day has had a gr eat impact on my scientific career. Also, I commend my thesis committee members; Dr. Ste phen Dodd, Dr. David Cris well and Dr. Krista Vandenborne for their guidance and support thro ughout my graduate work. Specifically, the interactions with Dr. Criswell and Dr. Dodd ha ve shown me the quali ties that successful scientists and educators possess. I also extend my thanks to Dr. Vandenborne for presenting me the opportunity to be involved with an NIH T32 award. The award provided additional opportunities for travel to scientif ic meetings and workshops allowing me to meet leaders in the biological sciences. I also wish to recognize both past and present members of the Powers laboratory who have contributed to my success. Most importantly, I am thankful for the experiences shared with my wife and family throughout my life and career. 4

PAGE 5

TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................................11 CHAPTER 1 INTRODUCTION................................................................................................................. .13 2 LITERATURE REVIEW.......................................................................................................16 MV Induced Myofiber Injury .................................................................................................16 Characteristics of Myofiber Injury ..................................................................................16 Mechanisms of Diaphragmatic Dysfunction ...................................................................17 Atrophy .....................................................................................................................17 Proteolysis ................................................................................................................18 Structural Injury .......................................................................................................18 Oxidative Stress ........................................................................................................19 Summary .................................................................................................................................24 3 MATERIALS AND METHODS...........................................................................................27 Experiment 1: Animals ...........................................................................................................27 Animal Model Justification .............................................................................................27 Animal Housing and Diet ................................................................................................27 Experimental Design .......................................................................................................27 Animal Protocol...............................................................................................................28 Statistical Analysis ..........................................................................................................29 Experiment 2: Cell Culture .....................................................................................................29 Experimental Design .......................................................................................................29 Cell Protocol ....................................................................................................................29 Statistical Analysis ..........................................................................................................30 General Methods .....................................................................................................................30 Animal Measurements .....................................................................................................30 Histological ..............................................................................................................30 Biochemical ..............................................................................................................31 Cell Culture Measurements .............................................................................................33 4 RESULTS...................................................................................................................... .........37 Animal Characteristics ............................................................................................................37 5

PAGE 6

Redox Balance ........................................................................................................................37 HO-1 ................................................................................................................................37 Ferritin .............................................................................................................................37 4-HNE ..............................................................................................................................38 Total Glutathione .............................................................................................................38 Gamma GCS ....................................................................................................................38 Proteolysis ...............................................................................................................................39 Calpain I and II ................................................................................................................39 Caspase-3 .........................................................................................................................39 Cytoskeletal Protein Degradation ....................................................................................40 Diaphragmatic Atrophy ..........................................................................................................40 Cross-sectional Area ........................................................................................................40 Myogenic Cells .......................................................................................................................41 Cell Viability ...................................................................................................................41 HO-1 Protein ...................................................................................................................41 Total Glutathione .............................................................................................................41 4-HNE ..............................................................................................................................42 5 DISCUSSION................................................................................................................... ......53 Overview of Principal Findings ..............................................................................................53 Induction of HO-1 during MV Attenuates Diaphragmatic Oxidative Stress ..................53 Overexpression of HO-1 Reduces Overal l MV-induced Protease Activation ................54 Overexpression of HO-1 Retards MV-induced Diaphragmatic Atrophy ........................56 Induction of HO-1 in Myotubes Fails to Maintain Redox Balance during H2O2 Exposure ......................................................................................................................56 Conclusions and Future Directions .........................................................................................58 LIST OF REFERENCES ...............................................................................................................60 BIOGRAPHICAL SKETCH .........................................................................................................71 6

PAGE 7

LIST OF TABLES Table page 4-1 Body mass and vital characteristics of expe rimental animals. Values are expressed as mean SEM. No significant differences were detected in any of the variables measured (P>0.05). CON= control; 12M V= 12 hours mechanical ventilation; 12MVH= 12 hours MV with hemin; 12MVI; 12 hours MV with CrMPIX; 18MV= 18 hours mechanical ventilation; 18MVH= 18 hours MV with hemin; 18MVI= 18 hours MV with CrMPIX. ...................................................................................................43 7

PAGE 8

LIST OF FIGURES Figure page 2-1 Potential sources of oxidant production in the diaphragm during MV. Periods of MV may cause an increase in one or more of th e following: 1) xanthine oxidase; 2) nitric oxide synthase; 3) NADPH oxidase; 4) m itochondrial; and 5) HO-1. Through these mechanisms, MV may cause an increase in the level of oxidative stress and subsequent injury of diaphragm myofibers. .......................................................................25 2-2 Proposed mechanism of HO-1 induced cyt oprotection in the diaphragm during MV. An increase in available heme stimulates the induction of the HO-1 gene and protein thereby increasing the activity of the enzyme. Upon the degradation of heme, the by-products carbon monoxide (CO), biliverdin and iron will be released. We propose a cytoprotective effect from this action where a de crease in oxidative stress and proteolysis is observed in the diaphragm. ...................................................................26 3-1 Experimental animal design examining the role of HO-1 in the diaphragm during MV. Measurements from MV, MVInhibito r, and MVHemin were made vs. CON. We addressed Aims 1 and 2 by having three levels of MV at two different durations. ....35 3-2 Experimental design for myogenic cell culture experiments. Treatments included saline and hydrogen peroxide for control treatments and hydrogen peroxide, HO-1 inhibitor/saline; HO-1 inhibitor/hydrogen peroxide was used in conjunction with hemin treatments in order to determin e the role of HO-1 in redox regulation. .................36 4-1 Fold changes (versus control) of HO-1 protein content in diaphragm samples. All animals mechanically ventilat ed exhibited a significant increase in HO-1 protein. In addition, the combination of hemin and MV re sulted in an enhanced level of protein content compared to all samples measured. Y values are mean fold change SE. Significantly increased versus CON (P< 0.05). # Significantly increased versus 12MV and 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). Significantly increased versus 12M VH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. .................44 4-2 Fold changes (versus control) of ferritin protein content in diaphragm samples. MV and MVH animals exhibited a significant incr ease in ferritin protein. Y values are mean fold change SE. Significan tly increased versus Con (P<0.05). # Significantly increased versus 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. ......................................................................44 4-3 Fold changes (versus control) of 4-HNE accumulation in diaphragm samples. Y values are mean fold change SE. Signi ficant increases in 4-HNE accumulation in 12MV, 18MV, and 18MVI treatments versus CON (P>0.05). # Significantly increased versus 18MVH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. ..........................................................45 8

PAGE 9

4-4 Total glutathione concentrations in diaphragm samples. 12 and 18MV and 12 and 18MVI significantly decreased vers us CON. 12 and18MV and 12 and 18MVI significantly lower than MVH. Y values are mean SE. Significantly decreased versus Con. #Significantly increased versus 12MV and 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. .................45 4-5 Fold changes (versus control) of gamma-GCS levels in diaphragm samples. MVH groups significantly increased versus all groups Y values are mean fold change SE. Significantly increased versus CON (P <0.05). #Significantly increased versus 12MV and 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. ..................................................................................................46 4-6 Fold changes (versus control) of active calpain I protein in diaphragm samples. 12 and 18 MV, 12MVH, and 12 and 18MVI signi ficantly increased versus CON. Y values are mean fold change SE. Significantly increased versus CON (P<0.05). # 12MVI significantly increased versus 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. .................46 4-7 Fold changes (versus control) of act ive calpain II protein measurements in diaphragm samples. Y values are mean fo ld change SE. No significant difference versus Con. (P>0.05). CON= Control; MV= mechanically ven tilated; MVH= MV with hemin; MVI= MV with CrMPIX. ..............................................................................47 4-8 Fold changes (versus control) of active caspase-3 protein measurements in diaphragm samples. 12 and 18 hour MV, MVH, and MVI significantly increased versus CON. Y values are mean fold cha nge SE. Significantly increased versus CON (P<0.05). # Significantly increased versus 12MVH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. ........47 4-9 Fold changes (versus control) of 145kDa -II spectrin degradati on product levels in diaphragm samples. Y values are mean fold change SE. *18MV and 18MVI significantly increased versus CON (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX.........................................48 4-10 Fold changes (versus control) of 120kDa -II spectrin degradati on product levels in diaphragm samples. Y values are mean fold change SE. *12MV, 18MV and 18MVI significantly increased vers us CON (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. .................48 4-11 Type I fiber cross-sect ional area in the diaphragm following 18 hours of MV. MV and MVI significantly decreased versus CON and MVH. MVI significantly lower than MVH. Y values are mean SE. Significantly decreased versus CON. # Significantly decreased versus MVH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX.........................................49 9

PAGE 10

4-12 Type IIa fiber cross-sect ional area in the diaphragm following 18 hours of MV. MV, MVI significantly decreased versus CON and MVH. Y values are mean SE. Significantly decreased versus CON (P< 0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX.........................................49 4-13 Type IIb/x fiber cross-sectional area in the diaphr agm following 18 hours of MV. MV, MVI significantly decreased versus C ON and MVH. Y values are mean SE. Significantly decreased versus CON (P< 0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX.........................................50 4-14 Cell viability after tr eatment with hemin (150 M). Cells were treated with hemin and viability was assessed using the Trypan blue exclusion method at 0, 2, 4 and 6 hours following treatment. Y values are mean percent survival SE. No significant difference existed between control and any time period (P>0.05). ...................................51 4-15 Fold change (versus control) of HO-1 pr otein levels in C2C12 myotubes. Y values are mean fold change SE. Significantly increased versus CON and CON H2O2. # Significantly increased versus Hemin a nd Hemin H2O2 (P<0.05). CON= Control; CON H2O2= Control with H2O2; Hemin= Hemin; Hemin H2O2= Hemin with H2O2; Hemin/I= hemin with CrMPIX; Hemi n/I H2O2= Hemin/CrMPIX with H2O2.................51 4-16 Total GSH concentration in cultured C2C12 myotubes. Y values are mean SE. *Hemin H2O2, Hemin/I and Hemin/I H2O2 significantly lower versus CON. #Hemin H2O2 significantly lower than CON H2O2. Hemin H2O2 significantly lower versus H (P<0.05). CON= Control; CON H2O2= Control with H2O2; Hemin= Hemin; Hemin H2O2= Hemin with H2O2; Hemin/I= hemin with CrMPIX; Hemin/I H2O2= Hemin/CrMPIX with H2O2...................................................................................52 4-17 Fold change (versus control) of to tal 4-HNE measurements in cultured C2C12 myotubes. Y values are mean SE. No si gnificant differences detected in groups (P>0.05). CON= Control; CON H2O2= Control with H2O2; Hemin= Hemin; Hemin H2O2= Hemin with H2O2; Hemin/I= Hemin with CrMPIX; Hemin/I H2O2= Hemin/CrMPIX with H2O2................................................................................................52 10

PAGE 11

Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy OVEREXPRESSION OF HO-1 ATTENUATES MV-INDUCED ATROPHY OF THE DIAPHRAGM By Darin James Falk December 2007 Chair: Scott K. Powers Major: Health and Human Performance Mechanical ventilation (MV) is the primar y means of support used in patients with respiratory muscle failure. The removal of patients from MV is termed weaning and problems in weaning occur frequently. Im portantly, weaning difficulties ar e attributed to diaphragmatic weakness characterized by atrophy and contractile dysfunction. Ox idative stress contributes to MV-induced diaphragmatic weakness. However, the sequence of events leading to oxidative stress has not been fully elucid ated. A key stress sensitive enzyme, heme oxygenase-1 (HO-1), is rapidly induced in the diaphragm during MV. Paradoxically, HO-1 may function either as a proor antioxidant and the role that HO-1 plays in MV-induced oxidative stress in the diaphragm is unknown. To address this question we mechanically ventilated rats for 12 or 18 hours (MV) with subsets of animals that combined MV along with a HO-1 inducing agent, hemin (MVH); or MV with a HO-1 activity inhibitor, CrMPIX (MV I). Indices of oxidative stress, proteolytic activation, and atrophy were measured in the di aphragm following the expe rimental protocol. Our study reveals that hemin-induced elevation of HO-1 during MV provides protection against oxidative injury, proteolytic activation, a nd diaphragmatic atrophy. Specifically, hemin administration preserves glutathi one levels and prevents lipid peroxidation in the diaphragm during MV. Further, overexpre ssion of HO-1 decreased the MV -induced activation of calpain 11

PAGE 12

and caspase-3 in the diaphragm. Finally, the hemin-induced resistance to oxidative stress and protease activation in the diaphragm, led to an attenuation of diaphrag matic atrophy following 18 hours of MV. We conclude that HO-1 does not f unction as a pro-oxidant in the diaphragm but instead provides antioxidant and cyt oprotective properties during MV. 12

PAGE 13

CHAPTER 1 INTRODUCTION A variety of conditions including spinal cord injury, drug over dose, surgery and periods of unconsciousness render the pulmonary system unable to sustain blood gas homeostasis. Positive pressure mechanical ventilation (MV) provides an unmatched superiority in sustaining adequate alveolar ventilation in these patient populations (39). Unfort unately, removal from the ventilator (weaning) is inherently difficult, accounting for more than 40% of the total time spent on the ventilator (27). In fact, approximately 25% out of all those requiring MV experience weaning difficulties translating to billions of dollars per year spent in weaning procedures (61). In order to elucidate the potential mechan isms of MV-induced w eaning difficulties, our laboratory and others have highlighted the link between oxidative stress and diaphragmatic complications (15, 28, 42, 100, 119, 129). The genera tion of reactive oxygen species (ROS) in the diaphragm during MV has several targets within diaphragm fibers (proteins, lipids, DNA) and contributes to their dysfunc tion(129). This occurs within 6 hours following the initiation of MV and oxidation of the key contractile proteins actin and myosin has been reported (129). Additionally, oxidative stress promotes the activa tion of skeletal muscle proteases(85, 97). Pilot data from our laboratory indicate s that the calpain and caspase-3 proteases are critical oxidative stress sensitive components in the development of atrophy. Consequent ly, these events may contribute to the MV-induced d ecrease in the diaphragmatic sp ecific force production and crosssectional area of diaphragm fibers. In this regard, we have focused on heme oxyge nase-1 (HO-1) as a pot ential regulator of redox control in the diaphragm dur ing MV. HO-1 is an intracellula r enzyme localized primarily to the microsomal fraction of the cell and is consid ered to illicit antioxida nt properties (47). The enzyme catalyzes the rate-limiting step in the degradation of heme resulting in the generation of 13

PAGE 14

carbon monoxide, biliverdin and free iron (Fe 2+ ). Importantly, biliverdin is further reduced to bilirubin via biliverdin reductase exhibiting poten t antioxidant effects. While the induction of the iron sequestering protein ferritin is cl osely associated with HO-1 accumulation, the possibility of radical formation exists due to the release of iron (7, 9, 58, 77, 92, 110). Consequently, it is yet to be es tablished whether HO-1 functions as a proor antioxidant in the diaphragm during MV. Therefore, these experiments investigated the role HO-1 plays in maintaining redox balance and minimizing subse quent protease activation in the diaphragm during MV. Our experiments were designed to achieve the following specific aims. Specific Aim 1: To determine if pharmacological induction of HO-1 expression in the diaphragm protects against MV -induced oxidative damage. Rationale: Our work indicates that oxidative stress plays a prominent role in the development of diaphragmatic inju ry during MV. It is also known that oxidative stress increases HO-1 in a variety of cell types. In addition, prior induction of HO-1 affords protection in a variety of cell types against ensu ing periods of oxidative stress. Hypothesis : Increased HO-1 expression in the diaphragm will attenuate oxidative stress during MV. Specific Aim 2: To determine if elevated levels of HO-1 will protect against MV-induced activation of calpain and caspase-3 in the diaphragm during prolonged MV. Rationale: Preliminary data from our laboratory indicate the proteases, calpain and caspase-3 as critical initiators of the proteolytic cascade in th e diaphragm during MV. Further, it appears likely that MV-induced oxidative stress is a prerequisite for the ac tivation of calpain and caspase-3. However, it is unknown whether HO-1 can mediate protection against protease activation in skeletal muscle by maintaining redox homeostasis. 14

PAGE 15

Hypothesis: An increase of HO-1 in the diaphragm will attenuate MV-induced protease activation (i.e., calpain and cas pase-3) and protect against MV-induced diaphragmatic atrophy. Specific Aim 3: To investigate the dose-response relationship between HO-1 levels and protection against reactive oxygen species-induced oxidative injury in skeletal muscle myotubes. Rationale: In various cell types, hi gh levels of HO-1 confer protection against oxidative stress. However, it is unknown whether prior induction of HO-1 improves or exacerbates the effects of oxidative stress in skeletal muscle myotubes. By ut ilizing a murine C2C12 myogenic model we will determine the importance of HO-1 fo r cellular protection against oxidative stress. Hypothesis : HO-1 acts as an antioxidant in skeletal muscle and protects against oxidative injury. 15

PAGE 16

CHAPTER 2 LITERATURE REVIEW The diaphragm is the primary muscle of re spiration in mammals and is necessary for normal breathing. In this regard, it is clear any perturbation to the diaphragm will have a major impact on the overall health of the individual. B ecause MV is associated with respiratory muscle weakness and subsequent weaning difficulties, elucidating the mechanisms of diaphragmatic injury is critical. Defining the possible mechanisms of protection against MV-induced diaphragmatic injury plays a pi votal role in defining optimal therapeutic strategies in the intensive care unit. A multitude of factors can contribute to MV-i nduced diaphragmatic injury. In order to focus the scope of this section, the role of oxidative stress and its contribution to diaphragmatic weakness will be discussed. Therefore, the objectiv es of this review ar e to characterize the diaphragmatic injury associated with MV and then discuss the potential sources of oxidative injury. The second objective of this revi ew will discuss the role of th e enzyme responsible for the degradation of heme: heme oxygenase-1 (HO-1). Though HO-1 has been primarily purported in the literature to serve as an antioxidant it has th e potential to function as a pro-oxidant. Therefore, this section will elaborate on the evidence suppor ting both of these notions to discern whether HO-1 serves as a pr oor antioxidant. MV Induced Myofiber Injury Characteristics of Myofiber Injury Respiratory muscle failure is ch aracterized by periods of respiration where an individual is unable to maintain sufficient alveolar ventilation. MV is used in orde r to achieve adequate ventilation, however difficulties are often succumbed when cessation of ventilator use (weaning) 16

PAGE 17

is deemed appropriate(61). An overwhelming breadth of evidence indicates that weaning difficulties are attributed to MV induced changes within the diaphragm (2, 15, 21, 22, 28, 31, 32, 42, 60, 81, 86-88, 94, 95, 98-100, 119, 127, 129, 131). Curren tly, there is exte nsive data from four animal models (rats, rabbits, pigs a nd baboons) along with limited data from human research which have begun to define th e effects of MV on the diaphragm. Importantly, all models of MV have illustrated a wide array of deleterious effects on the diaphragm(2, 50, 60, 62, 86, 88, 94, 124) Specifically, rodent studies have observed decreased diaphragmatic performance during periods of MV lasting from 12-48 hours (15, 60, 86, 93-95, 98, 119). Until recently, it was uncl ear whether the diaphragmatic dysfunction resulting from MV was similar in human patients. This is no longer a mystery, due to the novel approach undertaken by Levine and colleague s. Their investigations examined the response of the human diaphragm between 18-72 hours of MV. They obser ved an approximate 40% decrease in crosssectional area across both fiber-types(62). Addi tional evidence from human investigations has shown a 50% drop in twitch transdiaphragmatic pressu re in those with diseases as well as small decreases in cross-sectional area in a neonatal population following MV(50, 124). Clearly, limited human evidence justifies the importan ce of using animal studies in defining the mechanisms causing dysfunction and atrophy of the diaphragm resulting in weaning failure. Mechanisms of Diaphragmatic Dysfunction Atrophy Atrophy of the diaphragm has been reported in virtually all animal experiments following MV (2, 14, 31, 60, 71, 72, 127). Although atrophy occurs in a variety of disuse models, MV is unique in that a swift induction of atrophy occurs (i.e. 12 hours) (60, 71, 100, 127). A decrease in protein synthesis (56), an in crease in protein degrad ation (16), or combination of the two can result in disuse mediated atr ophy. Our laboratory has observed a d ecrease in protein synthesis in 17

PAGE 18

as few as 6 hours following MV (99). Further, decreases in protein synt hesis-related signaling and depression of type I and IIx myosin heavy chain are evident during 12-24 hours of MV (70, 99). Nonetheless, our past fi ndings suggest the rapid development of diaphragmatic atrophy following MV is primarily due to an increase in proteolytic-related mechanisms (22, 71, 100). Proteolysis There are several systems that contribute to sk eletal muscle degradation during periods of oxidative stress, including the lysosomal, cal pain, caspase-3, and proteasome pathways(13, 85, 121). Importantly, the contributions of the lysosomal and proteasome pathways play a minor role in the initial cascade of skeletal muscle breakdown during di suse (reviewed in(85). However, elevated and sustained increases in intracellula r calcium can activate both calpain and caspase-3. Both of these proteases have been shown to play a major role in skeletal muscle regulation by cleaving myofilament and cytoskeletal-related prot eins (20). Importantly, one of the hallmarks of oxidative stress is disruption of intracellular calcium homeostasi s (reviewed in (40). Therefore, cellular redox disturbances could elevate calpain and caspase-3 ac tivities and play a significant role in the progression of prot eolysis and dysfunction of diaphr agm myofibers during MV (45). By controlling the redox state and limiting the proteo lytic cascade of events in the cell it may be possible to attenuate MV-induced diaphragmatic injury. Structural Injury Alterations in myofiber st ructure such as membrane and mitochondrial disruption, appearance of lipid vacuoles in the sarcoplasm, and cytostructural protein degradation have been reported in skeletal muscle disuse models (14, 94 ). Furthermore, similar finding have been noted in diaphragms from mechanically ventilated animals (120) but it is important to note that hindlimb muscles (i.e. inactive) from mechani cally ventilated animals do not exhibit these markers (71, 94). The occurrence of myofiber disruption is notewo rthy due to the close 18

PAGE 19

association of myofiber injury and contractile dysfunction (94). Although the exact mechanisms are still unclear, several possibilities do exist. The activation of calpains and/or caspase-3 during MV may be responsible for init iating the cascade of events associated with myofilament degradation (85). Recently several investigation have shown th at inhibition of calpain or caspase-3 during MV can decrease proteolysis, atrophy and contractil e dysfunction (69, 71). Another possible mechanism for myofiber disruption would include reloading of the muscle following inactivity. Reloading of hindlimb muscles following ex tended periods of disuse is associated with increased vulnerability to muscle fiber injury (78). Therefore, we carefully control the level of anesthesia during MV to prevent spontaneous breathing by animals which would elicit a reloading type effect on the diaphragm. Nonetheless, our laboratory has addressed this issue where animals were ve ntilated for 24 hours and then allowed to spontaneously breathe for 2 hours. This protoc ol did not induce furthe r contractile dysfunction, membrane damage, or mediation of infl ammatory related species (119). Oxidative Stress The role of oxidative stress dur ing skeletal muscle atrophy wa s demonstrated in the early 1990s through pioneering studies by Kondo(51-55). Since that tim e, the relationship between oxidative stress and myofiber injury has been well documented (reviewed in(59, 84, 85, 89). Oxidative stress can alter the structure, function and/or regula tion of lipids, proteins, and nucleic acids. Due to these features, it is necessary to reveal the sources of oxidants contributing to redox imbalances in the cell. Numerous pathways exist (Fig. 2-1) and may independently or cooperatively contribute to myocyte damage includi ng: 1) xanthine oxidas e; 2) nitric oxide synthase (NOS); 3) NADPH oxidase ; 4) mitochondrial; and 5) HO-1. Xanthine Oxidase Xanthine oxidase (XO) is pr oduced via sulfhydryl oxidation or proteolysis of xanthine dehydroge nase by calcium activated proteas es (calpain)(35, 38). In the 19

PAGE 20

presence of oxygen and purine substrates (hypoxanthine, xanthine), XO catalyzes the formation of superoxide radicals and uric acid. Recent ev idence indicates that XO is found in skeletal muscle fibers in humans and other animals and contributes to XO mediat ed oxidant production in the diaphragm during contraction(38, 104). A lthough, during resistive breathing where oxidant production is evident, it appears the XO pathway plays a minor role(106, 107). Therefore, future investigations concerning XO are warranted in order to determine its contribution to oxidant production in the diaphragm during MV. NAD(P)H Oxidase. NAD(P)H oxidases are membrane-associated enzymes that catalyze the one-electron reduction of molecular oxyge n using either NADH or NADPH as electron donors. Importantly, there is a non-phagocytic and non-mitochondrial NAD(P)H oxidase is found in both human and rodent skeletal muscle(43). This is pertinent to our model where the contribution of sepsis related oxidant production has been minimalized. Numerous factors can increase NAD(P)H oxidase activity in cells including the calcium-sensitive PKC-ERK1/2 pathway and it is possible this pathway play s a role in NAD(P)H oxidase activity in the diaphragm during prolonged MV (37). Our la boratory has found that inhibition of NAD(P)H oxidase (apocynin) lessens MV -induced oxidative stre ss and contractile dysfunction in the diaphragm (VanGammeren et al). Nitric Oxide Synthase. Three isoforms of NOS exist( 44, 102): 1) inducible NOS (iNOS) is calcium independent; 2) endot helial NOS (eNOS) is calcium activated; and 3) neuronal NOS (nNOS) is also calcium activated. Both nNOS and eNOS are expressed in the diaphragm (102, 118) and iNOS is present during periods of endotoxin stress(11). Endogenous nitric oxide production via NOS can result in the forma tion of reactive nitroge n species, including peroxynitrite (ONOO ). Reactive nitrogen species are associ ated with cellular injury including 20

PAGE 21

mitochondrial dysfunction, lipid peroxidation, and nitrosylation of proteins (11, 79, 102, 108). Although, recent work from our lab suggests the contribution of n itric oxide to diaphragmatic dysfunction and injury is minor if even detectable during MV (118). Mitochondrial oxidants. The mitochondria have the capability to be a potent source of ROS production in the cell although (17, 84) (1). While the electron tran sport chain primarily functions to produce ATP, however, an electron leak may occur where side reactions produce superoxide, which is further dismutated to hydr ogen peroxide. In addi tion, due to the abundant production of oxidants in the mitochondrial matr ix and cytosol, the outer membrane also contributes to hydrogen peroxide generation. Clearly, the mitochondria have the pote ntial to shift cellular redox balance and during periods of sepsis and their role in cellular injury has been demonstrated in the diaphragm (109). However, the role of mitochondria l generated oxidants in the diaphragm is unclear in sepsis-free conditions where negligible effects were observed after 5 days of MV(30). Heme Oxygenase-1. The observation of the endogenous colored pigments biliverdin and bilirubin formed via the degradation of he me have been known for over 50 years but the enzymatic reactions responsible were not di scovered until many years later(113-115). Tenhunen et al. was first to illustrate th e effects of the enzyme, heme o xygenase, which is responsible for the degradation of heme (Fe-protoporphyrin-IX) and its production of biliverdin and finally bilirubin (114). There are two known isoforms of heme oxygenase which exist in the diaphragm, HO-1 and HO-2 (12, 112). Of the two isoforms, HO-1 has been shown to play a critical role in redox balance involving skeletal muscle (12, 41, 109) On the contrary, there is limited evidence showing a major role for HO-2 in skeletal muscle (12). Therefore, our focus has been conserved for HO-1 on cellular redox balance in these experiments (Fig 2-2). 21

PAGE 22

Various preparations including h eavy metals, endotoxin, inflammatory cytokines, hydrogen peroxide (H 2 O 2 ), quinones, and radiation (ultraviolet A) all have been shown to induce HO-1. Importantly, this provides protection wh ere upon inhibition of HO-1 exacerbates cellular injury (3, 12, 46-48, 57, 109). The induction of HO-1 depends highly on transcriptional activation of the HO-1 gene and the synthesis of mRNA regardless of cell type. Numerous observations have concluded that pretreatment with a HO-1 inducing agent can buffer cellular stress including inflammatory and cardiovascular diseases, lung injury, ischemia/reperfusion and organ transplantation (reviewed in(91). The protective effect of HO-1 is believed to function by the action of bilirubin where it has been shown to effectively quench singlet oxygen, scavenge hypochlorous acid, and inhibit lipid peroxidation. Bilirubin protects against cytotoxicity induced by H 2 O 2 and/or enzymatically generated ROS in endothelial cells, vascular sm ooth muscle cells, and cardiac myocytes (19, 76, 126). In fact, Baranano et al. found th at bilirubin protects against H 2 O 2 -mediated cell death at a 10,000 fold molar ratio of oxidant to antioxidant suggesting even a higher buffering capacity provided by the major cellular antio xidant glutathi one (10). Recent work characterizing the gene/pro moter regulation of HO-1 has uncovered additional protective roles of th e enzymes induction. These experi ments describe the regulation of the antioxidant response element (ARE) whic h is responsible for the expression of HO-1 and other antioxidant enzymes. In particular, -glutamylcysteine synthetase, NAD(P)H quinone reductase, glutathione S-transferase, thioredoxin, and ferritin light/heavy ch ains are all regulated through ARE induction (36, 49, 123). These enzymes have all been implicated in improving the redox capacity of cells to maintain cellular homeostasis. 22

PAGE 23

Contrary to the overwhelming support for HO-1 and maintenance of cellular redox control, there is also evidence for potenti al deleterious effects. The possibility exists where HO-1 acts as a pro-oxidant due to the release of reactiv e iron during the degrad ation of heme(57, 92). Transitional metal ions such as iron are capable of converti ng superoxide and H 2 O 2 to the highly reactive hydroxyl radical ( OH ) (shown below). Fe +2 + H 2 O 2 intermediates Fe +3 + OH + OH O 2 ._ + H 2 O 2 (with reactive iron or copper) O 2 + OH + OH Iron dependent radical production can function as a potent mediator of skeletal muscle injury (29, 73), Although the of and possible injury from thes e reactive iron exists, another positive feature of increased HO-1 is associated w ith the release of iron (7, 25, 26). In a variety of in vitro preparations the iron binding protein ferritin elicits cyt oprotective properties (7, 8, 68, 122). Ferritin sequesters free in tracellular iron where it can accommodate an ~ 1:4500 molar ratio(91). In this sense, the release of iron as a consequence of HO-1 activity may actually contribute to the protective mechan isms associated with HO-1 (122) The possibility of free iron mediated cellular damage via induction of HO1 exists but is not substantially supported. Apart from the well-defined function of HO1 in the breakdown of heme, it is now generally accepted as a mediator of cellular protection. Applications are being explored clinically where utilization of bile pigments have been effective in decreasing organ ischemia/reperfusion injury and transplant rejection (91). Moreove r, CO administration has been effective in alleviating the effects of inflammation, hypert ension, organ transplant ation, and vascular injury(91). Thus, the ther apeutic strategies that are currently under inves tigation are defining the protective properties of HO-1. To date, there have been only thr ee investigations examining the role of HO-1 in the diaphragm (12, 109, 112). Thes e experiments proved HO-1 to be critical in 23

PAGE 24

preserving diaphragmatic contractile and mitoc hondrial function in addition to maintaining redox buffering capacity during periods of inflamma tion. However, it is still unclear whether modulation of the HO-1 enzyme during MV func tions in a proor antioxidant manner. Therefore, elucidating the role of HO-1 is of keen interest in understanding redox homeostasis of the diaphragm during ventilation. Summary Through extensive research, our laboratory has demonstrated the use of MV as a model for studying skeletal muscle disuse and dys function of the diaphr agm (15, 21-23, 28, 70, 71, 86, 98-100, 118, 119, 129). By keeping an aseptic setti ng and maintaining blood gas homeostasis, MV serves as an excellent model for examini ng the mechanisms contributing to diaphragmatic injury and failure. Our lab has illustrated the effects of oxidativ e stress, proteolysis, and atrophy on diaphragm myofiber function during various periods of MV. However, it is still unknown which oxidant pathway(s) are re sponsible for these observations. Examining the role of HO-1 during MV will provide clarification of poten tial mechanisms causing complications in the diaphragm. Importantly, if redox balance can be maintained during MV, it is possible that rates of protease activity (calpain and caspase-3) may also be attenuat ed. In this regard, HO-1 may not only preserve redox status but may also limit the degree of atrophy and dysfunction of the diaphragm. 24

PAGE 25

Figure 2-1. Potential sources of oxidant production in the dia phragm during MV. Periods of MV may cause an increase in one or more of the following: 1) xanthine oxidase; 2) nitric oxide synthase; 3) NADPH oxidase; 4) mitoc hondrial; and 5) HO-1. Through these mechanisms, MV may cause an increa se in the level of oxidative stress and subsequent injury of diaphragm myofibers. 25

PAGE 26

Figure 2-2. Proposed mechanism of HO-1 induced cytoprotection in the diaphragm during MV. An increase in available heme stimulates the induction of the HO-1 gene and protein thereby increasing the activity of the enzyme. Upon the degradation of heme, the byproducts carbon monoxide (CO), biliverdin a nd iron will be released. We propose a cytoprotective effect from this action where a decrease in oxidative stress and proteolysis is observed in the diaphragm. 26

PAGE 27

CHAPTER 3 MATERIALS AND METHODS This chapter will be divided into two se gments. The first segment contains the investigative designs used in each of our experi ments that are intended to determine if HO-1 acts as a proor antioxidant in th e diaphragm during MV. In a subs equent section, we will provide the methodological details associated with each experimental protocol and measurement technique. Experiment 1: Animals Animal Model Justification Adult (4-6 month old) female Sprague-Dawley (S D) rats were used for experiment I. The animals were 4-6 months of age (young adult) at th e time of sacrifice. The SD rat was chosen due to similarities with the human diaphragm in anatomical and physiological parameters and the size of the animal also permits periodic arterial blood sampling (5, 6, 74, 75, 82, 83, 130). Animal Housing and Diet All animals were housed at the University of Florida Animal Care Services Center according to guidelines set forth by the Institutional Animal Care and Use Committee. Animals were maintained on a 12:12 hour light-dark cycle and provided food (AIN93 diet) and water ad libitum throughout the experimental period. Experimental Design In experiment I, adult rats (Harlan County) were randomly assigned to one of the following groups: 1) acutely anesthetized control (n = 8), 2) 12 hours of MV (n=8), 3) 12 hours of MV with CrMPIX (n = 8), 4) 12 hours of MV with hemin (n=8), 5) 18 hours of MV (n=8), 6) 18 hours of MV with CrMPIX (n=8), and 7) 18 hours of MV with hemin (n=8) (Figure 3-1). The Animal Care and Use Committee of the University of Florida has approved these experiments. 27

PAGE 28

Animal Protocol Animals in the control group were acutely an esthetized with sodi um pentobarbital (60 mg/kg IP). After reaching a surgical plane of an esthesia, the control animals were sacrificed immediately and the costal diaphragms were quickly removed and stored at C for subsequent analyses. Animals in the MV group were acutely anesth etized with sodium pentobarbital (60 mg/kg IP). After reaching a surgical plane of anesthes ia, the animals were tracheostomized utilizing aseptic techniques and mechanically ventilated with a controlled pressure-driven ventilator (Seimens) for 12 or 18 hours with the following settings: upper airway pressure limit: 20 cmH 2 O, pressure control level above PEEP: 4-6 cmH 2 O, respiratory rate: 80 bpm; PEEP: one cmH 2 O (15, 119). We have chosen 12 and 18 hours of mechanic al ventilation in orde r to establish a time course for these measurements because these periods of MV are associated with diaphragmatic contractile dysfunction, myofiber atrophy, increased oxidative stre ss and rates of proteolysis. Animals in the MVH group received an i.p. in jection of hemin (50mg/kg) 24 hours prior to the initiation of MV. For the MVI group, animals received a 5m i.p. injection of CrMPIX immediately prior to MV. Animals in the MV group received an i.p. injection of saline and surgical preparation, procedures, and animal mon itoring were performed as previously described (28). Following the termination of each expe rimental group the animals were immediately sacrifice and the costal diaphragm wa s quickly removed and stored at C for subsequent analyses. 28

PAGE 29

Statistical Analysis Group sample size was completed using a power analysis of preliminary data from our laboratory. Comparisons between groups were made by a 1 way ANOVA and, when appropriate, a Tukey HSD test was performed. Signi ficance was established at P < 0.05. Experiment 2: Cell Culture The myogenic cell line was chosen due to th e similarities between C2C12s and rodent skeletal muscle in cellular signaling and protein regulati on (24, 33, 63-66). Cells were maintained in a temperature and gas contro lled incubator throughout the experiments. Experimental Design In experiment II, cells were placed in one of the following groups: 1) control, 2) control/ H 2 O 2 3) hemin, 4) hemin/ H 2 O 2 5) hemin/inhibitor, and 6) hemin/inhibitor with H 2 O 2 (Fig 3-2). Cell Protocol Myogenic cells were cultured according to Li(67). Briefly, myotubes were cultured in DMEM supplemented with 10% fetal bovine serum and gentamicin at 37C in the presence of 5% CO 2 Myoblast differentiation was initiated by replacing the growth medium with differentiation medium supplemented with 2% horse serum. Differentiation was allowed to continue for 96 hours before experimentation, ch anging to fresh media every 48 hours. At the time of harvest, myotubes were washed 3x in PBS buffer (pH 7.4) before the addition of cell lysis buffer. After addition of lysis buffer, m yotubes were collected and centrifuged at 800g for 10min at 4 C. The supernatant was collected and stored at 80 C for subsequent analyses. In the hemin group, myotubes were culture d in horse serum combined with hemin (150 M) and were incubated for 4 h ours prior to harvest. Myotube s in the inhibitor group were grown in horse serum combined with CrMPIX (5 M) for 4 hours. Finally, myotubes in the 29

PAGE 30

hemin/inhibitor group were grown in horse serum with hemin (150 M)/inhibitor (5 M) for 4 hours. At the time of harvest, myotubes were collected as described above. We chose 100 M H 2 O 2 for the oxidative challenge in these experiments. This concentration of H 2 O 2 has been used in the literature in order to induce a stress response in C2C12 myotubes. In addition, this c oncentration may have relevance to in vivo cellular levels. Therefore, we chose this concentration to i nduce oxidative stress in the C2C12 protocol. H 2 O 2 treatment was administered for 2 hours following incubation with groups containing saline, hemin, or hemin/inhibitor. Following incubation with H 2 O 2 myotubes were collected as described above. Statistical Analysis Group sample size was completed using a power analysis of preliminary data from our laboratory. Comparisons between groups were made by a 1 way ANOVA and, when appropriate, a Tukey HSD test was performed. Signi ficance was established at P < 0.05. General Methods Animal Measurements Histological Immunohistochemistry. Diaphragm samples were remove d and fixed in OCT and stored at C. On the day of analysis, diaphragm sections (8 microns) from all groups were obtained and allowed to air dry at 25 C for 30 minutes. The slides were fixed in acetone (4 C) for 5 minutes and washed in separate 2 minute wash es in phosphate buffered saline. Serum blocking was performed on sections with incubation of normal goat serum (5%) blocking solution for 30 minutes. Primary HO-1 antibody incubation was diluted at 1:1000 in dystrophin solution for 60 minutes at 25 C. This was followed by an additional wash in PBS buffer with rhodamine for 30 30

PAGE 31

minutes at 25 C. Secondary antibody incubation was done at a concentration of 1:100 (goat anti-mouse IgG) with Rhodamine for 30 min at 25 C. A final rinse in PBS-T (3x) at 2 minute intervals was performed on all s ections. Slides were then mo unted in fluorescent mounting medium (Vector Laboratories) and images were acquired via a monochrome camera (Qimaging Retiga) attached to an inverted fluo rescent microscope (Axiovert 200, Xeiss). Myofiber Cross-Sectional Area. Sections from frozen diaphr agm samples were cut at 10 (microns) using a cryotome (Shandon Inc., Pitt sburgh, PA) and stained for dystrophin, myosin heavy chain (MHC) I and MHC type IIa proteins for fiber cross-sec tional area analysis (CSA) as described previously (71). CSA was determ ined using Scion software (NIH). Biochemical Heme Oxygenase-1 Activity. Heme Oxygenase-1 is the i nducible isoform of Heme Oxygenase and catalyzes the reaction of NADPH and O 2 for oxidation of heme producing CO, ferrous iron and biliverdin. The bile pigment biliverdin is reduced to bi lirubin via biliverdin reductase and both biliverdin and bilirubin ar e known to possess antioxidant properties (103, 117). HO activity in the microsomal fraction of diaphragm homogenate was assessed according to Gonzalez with modifications(34). Briefly, diaphragms were homogenized using a Polytron homogenizer in a 30 mM Tris-HCL buffer c ontaining 0.25 M sucrose; 0.15 M NaCl; 1mM DTT and a commercially available protease inhibitor co cktail (Sigma, St. Louis, MO). After brief sonication, crude homogenate was cen trifuged at 10,000g for 15 min at 4 C and the subsequent supernatant fraction centrifuge d at 100,000g for 60 minutes at 4 C. Microsomal fractions were resuspended in 0.1 ml of 100mM potassium phos phate buffer (pH 7.4), containing 2 mM MgCl2. Protein concentration was determined using th e Bradford method. HO-1 activity was assessed by combining the microsomal fraction (1 mg), biliverdin reductase (2 mg), 100mM potassium 31

PAGE 32

buffer (pH 7.4), containing 2mM MgCl2, 10 M hemin, 2mM glucose 6-phosphate, 0.2 U glucose 6-phosphate dehydrogenase, and 0.8 mM NADPH. Samples were incubated for 60 minutes at 37 C and terminated by the addition of 1 mL chloroform (Sigma, St. Louis, MO). The amount of extracted bilir ubin was calculated by the differe nce in absorption between 464nm and 530nm. Western Blot Analysis. Protein content was determined in all samples via Western Blot analysis. Diaphragm samples were homogenize d in a buffer containi ng: 30mMTris-HCL (pH 7.5), 250mM Sucrose, 150mM NaCl, 1mM DTT, a nd protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 4 C for 12 minutes at 16000g. After centrifugation, the supernatant was collected and a protein assay (Bradford) was pe rformed. Proteins from the supernatant fraction were separated via polyacr ylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with prim ary antibodies directed against the protein of interest. 4-HNE was probed as a measurement i ndicative of oxidative stress while HO-1, ferritin and gamma-GCS were performed to assess redox balance. Proteolytic activity was assessed by analyzing both total (non-active) and cleaved (active) calpain I and caspase-3. In addition, calpain II, calpastatin and -II spectrin were measured to obtain a more defined picture of calpain regulation during MV. Blots were imaged using an Odyssey system (Li-Cor Biosciences), using direct infrared fluorescent de tection with a wide linear dynamic range. Nuclear and Cytosolic Fractionation. Fractionation of diaphragm tissue was performed according to Alway et al(101). Briefly, diaphragm samples were homogenized in Buffer A containing 10mM NaCl, 15mM MgCl2 and 20 mM Hepes, Glycerol 20%, Triton X-100 0.1%, 1mM DTT and protease inhibitor cocktail (Sigma). Diaphragm homogenate s were centrifuged at 880g for 3 min at 4 C. Supernatants (cytosolic fraction) were collecte d and the pellet was saved 32

PAGE 33

for nuclear fractionation. Cytosolic prepar ation was performed by centrifugation of the supernatant at 3500g for 5min at 4 C and this was repeated 3 times by collecting the supernatant after each spin. Nuclear prepara tion was performed by washing the nuclear pellet with Buffer A and centrifugation at 880g for 3 min at 4 C. This step was repeated 3 times and pellets were the resuspended in Buffer A containing 41.5l of 5 M NaCl. Resuspended nuclear pellets were rotated for 60 minutes at 4 C. Samples were centrifuged at 21,900g for 15min at 4 C after rotation and the final supernatant was collect ed, as it is the nuclear protein fraction. Total Glutathione. Diaphragm tissue was assayed for to tal glutathione content using a commercially available kit according to the manuf acturers instructions (Cayman Chemical, Ann Arbor, MI). Cell Culture Measurements Trypan Blue Exclusion Assay. The trypan blue assay is used to assess cellular viability upon exposure to experimental treatments. Briefl y, an aliquot of cell suspension was diluted 1:1 with 0.4% trypan blue and cells we re counted with a hemocytomete r. Results are expressed as the percentage ratio of viable (unstained ) cells in treated vs. untreated samples. Western Blot Analysis. Protein content was determined in all samples via Western Blot analysis. Myotubes were lysed in a buffe r containing: 30mM Tris-HCL (pH 7.5), 250mM Sucrose, 150mM NaCl, 1mM DTT, and protease inhibitor cocktail (Sigma, St. Louis). Lysates were centrifuged at 4 C for 12 minutes at 16000g. After centrifugation, the supernatant was collected and a protein assay (Bradford) was perf ormed. Proteins from the supernatant fraction were separated via polyacrylamide gel electrophores is, transferred to a nitrocellulose membrane, and incubated with primary antibodies directed against the protein of interest. 4-HNE adduct accumulation was probed as a measurement indica tive of oxidative stress. HO-1 was probed to 33

PAGE 34

verify HO-1 induction during the experiments. Blots were imaged using an Odyssey system (LiCor Biosciences), using direct infr ared fluorescent detection with a wide linear dynamic range. Total Glutathione. Diaphragm tissue was assayed for to tal glutathione content using a commercially available kit according to the manuf acturers instructions (Cayman Chemical, Ann Arbor, MI). 34

PAGE 35

Figure 3-1. Experimental animal design examini ng the role of HO-1 in the diaphragm during MV. Measurements from MV, MVInhibito r, and MVHemin were made vs. CON. We addressed Aims 1 and 2 by having three le vels of MV at two different durations. 35

PAGE 36

Figure 3-2. Experimental design for myogenic cell cultu re experiments. Treatments included saline and hydrogen peroxide for control treatments and hydrogen peroxide, HO-1 inhibitor/saline; HO-1 inhibitor/hydrogen peroxide was used in conjunction with hemin treatments in order to determine the role of HO-1 in redox regulation. 36

PAGE 37

CHAPTER 4 RESULTS Animal Characteristics The physical and vital characteristics record ed for animals during all experiments are presented in Table 4-1. No si gnificant differences in animal body weight, heart rate, or blood pressure existed between experimental groups. Redox Balance HO-1 Cellular protection mediated via HO-1 inducti on is thought to reside through action of bilirubin where it has been shown to effectiv ely quench singlet oxygen, scavenge hypochlorous acid, and inhibit lipid peroxida tion. Bilirubin protects against cytotoxicity induced by H 2 O 2 and/or enzymatically generated ROS in endothe lial cells, vascular smooth muscle cells, and cardiac myocytes. Compared to control, HO-1 protein levels in the diaphragm were significantly increased in all MV treatments (Figure 4-1). Moreover, MVH treatment resulted in a 4.5 fold further increase in diaphragmatic HO-1 levels at 12 hours and ~6 fold in crease at 18 hours when compared to MV treatment alone. Thus, prol onged MV induces HO-1 protein expression in the diaphragm and when combined with the HO-1 in ducer hemin, there is a synergistic effect. Ferritin In a variety of in vitro preparations the iron binding prot ein ferritin elicits cytoprotective properties during periods of oxidative stress. Ferritin sequesters free intracellular iron and therefore increased ferritin content in cells would attenuate th e oxidant generating potential of reactive iron. In 12 and 18 hour MV experiments, both the MV and MVH treatments resulted in a significant increase in ferritin protein content in the diaphragm (Figure 4-2). In contrast, the MVI treatment did not result in a significant rise in diaphragmatic ferritin protein following 12 or 37

PAGE 38

18 hours of MV. This finding could be due, at least in part, to the fact that inhibition of HO-1 activity would retard the release of free iron and ther efore lower the stimulus to express ferritin. 4-HNE 4-HNE is an unsaturated hydroxyalkenal that is generated during the lipid peroxidation cascade. Furthermore, 4-HNE is the primar y adduct formed during this process and is commonly used to assess protein damage during oxidative stress. In 12 hour MV experiments, a significant increase in diaphragmatic 4-HNE was observed in the MV group only with no change in diaphragmatic levels of 4-HNE in the MVI group. However, in 18 hour experiments, both MV and MVI treatments result in a significant increase in 4-HNE accumulation in the diaphragm whereas diaphragmatic 4-HNE levels in the MVH animals were maintained at basal levels (Figure 4-3). Thus, it appears hemin-induced expression of HO-1 in the diaphragm assists in maintenance of redox balance during MV and atte nuates the degree of oxi dative cellular injury. Total Glutathione Glutathione is the major nonezymatic antioxidant component of the cell. It is present in mM concentrations and depletion of cellular stores is generally consid ered indicative of oxidative stress. Following 12 and 18 hours of MV there was a significant depletion of total GSH in diaphragms of both the MV and MVI groups. Conversely, the a ddition of hemin during 12 and 18 hour MV results in GSH preserva tion in the diaphragm (Figure 4-4). Gamma GCS Gamma-GCS is the rate-limiting enzyme required for glutathione synthesis. Measurements of this enzyme were performed to determine if our experimental treatments influenced the regulation of glutathione synt hesis in the diaphragm. During 12 and 18hr experiments, animals in the MVH group exhibite d a significant increase in gamma-GCS content when compared to Con, MV and MVI groups (Figure 4-5). Thus, maintenance of glutathione 38

PAGE 39

content in MVH animals may be due, at least in pa rt, to an increase in gamma-GCS levels in the diaphragm. Proteolysis Calpain I and II The activation of the calcium activated neutra l proteases, calpain I and II has been shown to occur in skeletal muscle in several models of disuse atrophy Upon activation by elevated and sustained increases in intracellular calcium, cal pains are capable of de grading both intact and disassociated skeletal muscle proteins. During 12 hour experime nts (MV, MVH, and MVI), all of the experimental MV treatments resulted in a significant increas e in active calpain I in the diaphragm, as detected by wester n blots measuring the cleaved (a ctive) 76kDa calpain fragment (Figure 4-6). However, following 18 hours of MV, diaphragms from MVH animals do not show a significant increase in active calpain I, while calpain I activities in the diaphragms from animals in the MV and MVI trea tments are still elevated. Fina lly, calpain II activity in the diaphragm was not altered in any groups at 12 or 18 hours when compared to control (Figure 47). Caspase-3 The cleavage of procaspase-3 results in the ac tivation of caspase-3. On ce active, caspase-3 can independently participate in the breakdown of intact myofibrillar proteins. Therefore, we evaluated the activity of this prot ease to assess its involvement in the cleavage of myofibrillar and cytoskeletal proteins in the diaphragm during MV. Utilizing immunoblotting techniques our results indicate that in all MV treatments (MV, MVH, and MVI) diaphragmatic caspase-3 acitivity was significantly elevated at 12 and 18 hours (Figure 4-8). 39

PAGE 40

Cytoskeletal Protein Degradation -II spectrin. -II spectrin is a cytoskeletal structural protein present in skeletal muscle. During periods of increased proteolytic activity, -II spectrin exhibits signature cleavage products that can be used to detect cleavage by both calpain and caspase-3. Specifically, the intact form of -II spectrin exists as ~250kDa protein and upon degradation yields a 145 kDa (calpain specific) and 120kDa (caspa se-3) cleaved bands. Thus, probing -II spectrin cleavage products can be used to determine in vivo calpain and caspase-3 activit ies. Twelve hours of MV resulted in a significant increase in diaphragmatic levels of cleaved 120kDa -II spectrin in the MV group (Figure 4-10). However, following 18 hours of MV, there was a significant increase in both calpain and caspase-3 specific degradation products (i.e. 145kDa and 120kDa bands, respectively) -II spectrin in diaphragms of both MV and MVI groups (Figures 4-9 and 4-10, respectively). Diaphragmatic Atrophy Cross-sectional Area To evaluate the impact of MV with overexpr ession and inhibition of HO-1 activity in the diaphragm, we compared the cross-sectional ar eas (CSA) of diaphragma tic myofibers across our experimental groups. Compared to control, we observed a significant decrease in diaphragmatic type I fiber CSA in the MV and MVI groups fo llowing 18 hours of MV. In contrast, the MVH treatment reduced diaphragmatic atrophy as eviden ced by the finding that diaphragmatic type I, IIa, and IIb/x fiber CSA did not differ between control and MVH anim als. Although the mean cross sectional areas of type I, IIa, and IIx/b diaphragm fibers did not differ (P>0.05) between the control group and the MVH group, note that no significant differences also existed in diaphragm fiber CSA between the MV group and the MVH group. Nonetheless, type I fibers were significantly larger in diaphragms from M VH treated animals compared to the MVI group 40

PAGE 41

(Figure 4-11). Finally, significant decreases in CS A for type IIa and type IIb/x were observed in diaphragms from MV and MVI animals while no significant differences existed in fiber CSA between Con and MVH (Figure 4-12, 4-13). Myogenic Cells Cell Viability In our cell culture experiments, we performed cell viability measurements on C2C12 myotubes treated with hemin (150 M). These measurements were performed to determine if the hemin treatment was toxic to our cell line. Importa ntly, our results revealed that hemin treatment was not toxic to myocytes in our experimental model as indicated by the failure of myotubes to take up Trypan blue (Figure 4-14). HO-1 Protein Measurements were made to assess HO-1 prot ein content in our cultured myotubes to determine if there was a similar induction of HO-1 in cell culture compared to the in vivo animal experiments. When compared to CON, hemin tr eated cells exhibited an ~15 fold increase in HO-1 protein and cells treated with hemin and hemin/inhibitor showed ~22 fold increase in HO1 protein when compared to CON (Figure 4-15 ). The observation that compared to hemin treatment alone, cells treated with both hemin and the HO-1 inhibitor exhibited significantly higher levels of HO-1 protein may be explained by a negative feedback system where the expression of the enzyme is upregulated due to the inhibition of its activity in an effort to maintain the cells capacity to degrade heme. Total Glutathione Contrary to the findings in our animal experiments, cells treated with hemin did not exhibit a protective phenotype against oxidativ e stress when challenged with H 2 O 2 In fact, hemin treated cells exposed to oxidative via H 2 O 2 showed a significant decr ease in total glutathione 41

PAGE 42

42 when compared to hemin treatment alone (Fi gure 4-16). When comparing the experimental groups; hemin, hemin/ H 2 O 2 hemin/inhibitor, and hemin/inhibitor plus H 2 O 2 all showed a significant depletion of total glutathione when compared to control. Hence, these findings suggest that hemin-induced e xpression of HO-1 in C2C12 my otubes does not protect against H 2 O 2 induced depletion of glutathione. 4-HNE As a second marker of oxidative stress, we also measured the levels of 4-HNE in the experimental myotubes. Identi cal to our aforementioned glutat hione measurements in cells, compared to control, hemin treated myotubes were not protected against H 2 O 2 induced increases in cellular levels of 4-HNE (Figure 4-17). These resu lts do not support the notion that hemin-induced increases in HO-1 levels in myotubes protects these cells against H 2 O 2 -induced lipid peroxidation.

PAGE 43

43 Table 4-1. Body mass and vital characteristics of experimental animals. Values are expressed as mean SE. No significant differences were detected in any of the variables measured (P>0.05). CON= control; 12MV= 12 hours mechanical ventilation; 12MVH= 12 hours MV with hemin; 12MVI; 12 hours MV with CrMPIX; 18MV= 18 hours mechanical ventilation; 18MVH= 18 hours MV with he min; 18MVI= 18 hours MV with CrMPIX. Variable CON 12MV 12MVH 12MVI 18MV 18MVH 18MVI Weight (kg) 0.301.019 0.308.019 0.302.029 0.292.012 0.296.016 0.297 .025 0.304 .014 Heart Rate (BPM) 357.6.4 370.4.0 348.3.4 354.1.2 350.7.9 352.9.1 Blood Pressure (mmHg) 96.9.5 101.3.7 97.9.6 76.1.4 79.8.7 83.5.4

PAGE 44

Figure 4-1. Fold changes (versus control) of HO-1 protein cont ent in diaphragm samples. All animals mechanically ventilat ed exhibited a significant increase in HO-1 protein. In addition, the combination of hemin and MV re sulted in an enhanced level of protein content compared to all samples measured. Y values are mean fold change SE. Significantly increased versus CON (P<0.05) # Significantly increased versus 12MV and 12MVI (P<0.05). Significantly increased vers us 18MV and 18MVI (P<0.05). Significantly increased versus 12M VH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. Figure 4-2. Fold changes (versus control) of ferr itin protein content in diaphragm samples. MV and MVH animals exhibited a significant incr ease in ferritin protein. Y values are mean fold change SE. Significan tly increased versus Con (P<0.05). # Significantly increased versus 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. 44

PAGE 45

Figure 4-3. Fold changes (versus control) of 4-HNE accumulation in diaphragm samples. Y values are mean fold change SE. Signi ficant increases in 4-HNE accumulation in 12MV, 18MV, and 18MVI treatments versus CON (P>0.05). # Significantly increased versus 18MVH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. Figure 4-4. Total glutathione concentrations in diaphragm sa mples. 12 and 18MV and 12 and 18MVI significantly decreased vers us CON. 12 and18MV and 12 and 18MVI significantly lower than MVH. Y values are mean SE. Significantly decreased versus Con. #Significantly increased versus 12MV and 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. 45

PAGE 46

Figure 4-5. Fold changes (versu s control) of gamma-GCS levels in diaphragm samples. MVH groups significantly increased versus all groups. Y values are mean fold change SE. Significantly increased versus CON (P <0.05). #Significantly increased versus 12MV and 12MVI (P<0.05). Significantly increased versus 18MV and 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. Figure 4-6. Fold changes (versus control) of active calpain I pr otein in diaphragm samples. 12 and 18 MV, 12MVH, and 12 and 18MVI signi ficantly increased versus CON. Y values are mean fold change SE. Significantly increased versus CON (P<0.05). # 12MVI significantly increased versus 18MVI (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. 46

PAGE 47

Figure 4-7. Fold changes (versus control) of active calpain II protein measurements in diaphragm samples. Y values are mean fo ld change SE. No significant difference versus Con. (P>0.05). CON= Control; MV= mechanically ven tilated; MVH= MV with hemin; MVI= MV with CrMPIX. Figure 4-8. Fold changes (versus control) of active caspase-3 protein measurements in diaphragm samples. 12 and 18 hour MV, MVH, and MVI significantly increased versus CON. Y values are mean fold cha nge SE. Significantly increased versus CON (P<0.05). # Significantly increased versus 12MVH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. 47

PAGE 48

Figure 4-9. Fold changes (versus control) of 145kDa -II spectrin degradati on product levels in diaphragm samples. Y values are mean fold change SE. *18MV and 18MVI significantly increased versus CON (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX. Figure 4-10. Fold changes (versus control) of 120kDa -II spectrin degradation product levels in diaphragm samples. Y values are mean fold change SE. *12MV, 18MV and 18MVI significantly increased vers us CON (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with hemin; MVI= MV with CrMPIX. 48

PAGE 49

Figure 4-11. Type I fiber cross-sectional area in the diaphragm following 18 hours of MV. MV and MVI significantly decreased versus CON and MVH. MVI significantly lower than MVH. Y values are mean SE. Significantly decreased versus CON. # Significantly decreased versus MVH (P<0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX. Figure 4-12. Type IIa fiber cr oss-sectional area in the diaphragm following 18 hours of MV. MV, MVI significantly decreased versus C ON and MVH. Y values are mean SE. Significantly decreased versus CON (P< 0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX. 49

PAGE 50

Figure 4-13. Type IIb/x fiber cross-sectional area in the diaphragm following 18 hours of MV. MV, MVI significantly decreased versus C ON and MVH. Y values are mean SE. Significantly decreased versus CON (P< 0.05). CON= Control; MV= mechanically ventilated; MVH= MV with he min; MVI= MV with CrMPIX. 50

PAGE 51

Figure 4-14. Cell viability af ter treatment with hemin (150 M). Cells were treated with hemin and viability was assessed us ing the Trypan blue exclusi on method at 0, 2, 4 and 6 hours following treatment. Y values are mean percent survival SE. No significant difference existed between control and any time period (P>0.05). Figure 4-15. Fold change (versus control) of HO -1 protein levels in C2C12 myotubes. Y values are mean fold change SE. Significantly increased versus CON and CON H2O2. # Significantly increased versus Hemin a nd Hemin H2O2 (P<0.05). CON= Control; CON H2O2= Control with H 2 O 2 ; Hemin= Hemin; Hemin H2O2= Hemin with H 2 O 2 ; Hemin/I= hemin with CrMPIX; Hemi n/I H2O2= Hemin/CrMPIX with H 2 O 2 51

PAGE 52

Figure 4-16. Total GSH concentration in cultur ed C2C12 myotubes. Y values are mean SE. *Hemin H2O2, Hemin/I and Hemin/I H2O2 significantly lower versus CON. #Hemin H2O2 significantly lower than CON H2O2. Hemin H2O2 significantly lower versus H (P<0.05). CON= Control; CON H2O2= Control with H 2 O 2 ; Hemin= Hemin; Hemin H2O2= Hemin with H 2 O 2 ; Hemin/I= hemin with CrMPIX; Hemin/I H2O2= Hemin/CrMPIX with H 2 O 2 Figure 4-17. Fold change (versus control) of total 4-HNE measurements in cultured C2C12 myotubes. Y values are mean SE. No si gnificant differences detected in groups (P>0.05). CON= Control; CON H2O2= Control with H 2 O 2 ; Hemin= Hemin; Hemin H2O2= Hemin with H 2 O 2 ; Hemin/I= Hemin with CrMPIX; Hemin/I H2O2= Hemin/CrMPIX with H 2 O 2 52

PAGE 53

CHAPTER 5 DISCUSSION Overview of Principal Findings These experiments tested the hypothesis that increased levels of HO-1, an endogenous enzyme with antioxidative properties, would re tard MV-induced oxida tive injury in the diaphragm. Our findings support this postulate as overexpression of HO-1 attenuated MVinduced oxidative stress in the diaphragm. We also predicted that MV-induced protease activation and atrophy of the diaphr agm would be diminished with elevated levels of HO-1. Our results also support this postulate as overexpr ession of HO-1 in the diaphragm retarded MVinduced calpain and caspas e-3 activity and myofiber atrophy in type I, IIa and type IIb/x fibers. Finally, to further investigat e the antioxidant role of HO1, we utilized a myogenic cell culture model to determine wh ether overexpression of HO-1 protects against ROS-mediated oxidative injury. Our results indicate that hemin-induced overexpression of HO-1 in myotubes does not protect these cells against oxidative injury induced by exposure to H 2 O 2 A detailed discussion providing an interpreta tion of our experiments follows in the subsequent sections. Induction of HO-1 during MV Attenua tes Diaphragmatic Oxidative Stress Prolonged MV is associated with a shift in th e pro-to-antioxidant balance in the diaphragm resulting in increased protein ox idation and lipid peroxidation in this key inspiratory muscle (120). The diaphragmatic endogenous antioxida nt system is intricate and involves both enzymatic and nonenzymatic antioxidants. In this regard, prolonged MV depletes diaphragmatic glutathione stores but also increases the expression of at l east two antioxidant enzymes, thioredoxin reductase-1 and manganese superoxide dismutase (28). Of furt her interest is that HO-1 protein abundance and gene ex pression rapidly increase in th e diaphragm during MV (28). However, there is uncertainty in the literature whether overexpression of HO-1 is beneficial or 53

PAGE 54

deleterious to cells due to its reported actions as e ither a proor antioxidant (92). In theory, HO1 can serve as a pro-oxidant by increasing the availability of free iron in the cell or as an antioxidant by producing both biliru bin and biliverdin. In an atte mpt to determine whether HO-1 functions as a proor antioxida nt in the diaphragm during MV, we independently elevated HO-1 levels in the diaphragm and in parallel experi ments we suppressed the activity of HO-1. Our findings support the concep t that HO-1 acts as an antioxidant in the diaphragm as hemin-induced overexpression of HO-1 was associated with redu ced oxidative stress during MV as indicated by the preservation of glutathione levels and the attenuation of lipid peroxidation. Moreover, pharmacological inhibition of HO1 activity was associated with the normal progression of MVinduced oxidative stress in the diaphragm, verify ing that HO-1 does not act as a pro-oxidant in this model. Overexpression of HO-1 Reduces Overa ll MV-induced Protease Activation It is well established that inactivity in skeletal muscles results in the activation of proteolytic pathways and that antioxidant ad ministration has been su ccessful in decreasing proteolysis (15, 72, 97). Therefore, we postulated that if HO-1 acts as an antioxidant in skeletal muscle, overexpression of HO-1 would diminish the activation of key protea ses (i.e., calpain and caspase-3) in the diaphragm during prolonged MV We focused our interest on calpain and caspase-3 because the induction of the calpains (i.e. I and II) a nd caspase-3 during periods of disuse may be a required first step to initiate the cascade of events underlying skeletal muscle atrophy (85, 111, 116). Indeed, our laboratory has show n that caspase-3 plays a critical role in skeletal muscle atrophy as pharmacological inhib ition of caspase-3 activit y retards diaphragmatic atrophy during 12 hours of MV (72). Furthermore, the administration of a calpain inhibitor (leupeptin) is also effective in attenuating diaphragmatic atro phy and contractile dysfunction during prolonged (i.e., 24 hours) MV (69). In th e present study, we assessed the influence of 54

PAGE 55

HO-1 levels on protease activation in two different ways. First, we assayed the activity of both calpain (I & II) and caspase-3 via western blotting to determine the level of activation in the diaphragm at the completion of our MV experime nts. Because this technique does not evaluate the contributions of calpain and capase-3 to muscle proteolysis in vivo, we also used an innovative and complimentary technique to asse ss calpain and caspase -3 activation in the diaphragm. This second technique is a novel method capable of measuring calpain and caspase-3 protease activity over time in muscle fibers. Briefly, this technique measures degradation of specific endogenous calpain and caspase-3 substrates (i.e., -II spectrin ) within muscle and provides an index of in vivo calpain and caspase-3 activity in the diaphragm over prolonged periods of MV. Using the direct approach measuring calpa in I activity, our results reveal that overexpression of HO-1 is associated with reduced calpain I activation in the diaphragm of MV animals. In contrast, overexpression of HO-1 did not appear to influence diaphragmatic caspase3 activity at the completion of 12 or 18 hours of MV Note, however, that these activity assays are single measurements (i.e. snapshot) of calpain and caspase-3 activity at one point in time (i.e., end of experiment). Therefore, these measur es fail to provide an accurate assessment of in vivo activity levels in the diaphragm over time. A mo re valid measurement of calpain and caspase-3 activity during MV ma y be achieved by assessment of specific calpain and caspase-3 degradation products of -II spectrin. Indeed, the accumulation of signature cleavage products of -II spectrin represent the specific activity of both calpain and caspase-3. In hemin-treated animals overexpressing HO-1, a reduction in the appearance of calpain specific (145kDa) and caspase-3 specific ( 120kDa) cleavage of -II spectrin breakdown products were observed in the diaphragm following 18 hours of MV. These obser vations suggest that HO-1 overexpression is 55

PAGE 56

associated with a decrease in the proteolytic-me diated damage to structural components within the diaphragm during MV. These findings, in conjunction with previo us results from our laboratory, suggest that increasi ng the diaphragmatic antioxidant capacity attenuates protease activation and may be effective in the preservation of diaphrag matic properties (i.e. atrophy and contractile dysfunction) during MV (15, 72). Overexpression of HO-1 Retards MV-induced Diaphragmatic Atrophy Based upon our finding that overexpression of HO-1 was associated with decreased oxidative stress and attenuated protease activatio n in the diaphragm, we anticipated that hemininduced expression of HO-1 would significantly retard MV-induced diap hragmatic myofiber atrophy. Our results support this notion as hemi n-induced expression of HO-1 protected against MV-induced atrophy in type I, type IIa, and ty pe IIb/x fibers. Nonetheless, although the mean CSA of type I, IIa, and IIb/x diaphragm fibers did not differ (P>0.05) between the control group and the hemin-treated MV group, no differences al so existed in diaphragm fiber CSA between the MV group and the hemin-treated MV group. We interpret this finding as an indication that while the overexpression of HO-1 diminished MV -induced myofiber atrophy, elevated levels of HO-1 did not completely prevent MV-induced diaphragmatic fiber atrophy. Induction of HO-1 in Myotubes Fails to Maintain Redox Balance during H 2 O 2 Exposure Oxidant challenges (H 2 O 2 ) to C2C12 myotubes can induce oxid ative stress, proteolysis and myotube atrophy (65, 105). Therefore, as a means of providing a controlled in vitro environment to study the antioxidant potential of HO-1 to protect cells against a H 2 O 2 challenge, we included a myogenic cell culture model in our experiments. Specifically, we tested the hypothesis that hemin induced increases in HO-1 levels in myotubes would provide antioxidant protection against an oxidative insult (100 M H 2 O 2 ). Our results indicate that hemin induced an increase in HO-1 protein expression and di d not alter myotube viabilit y. However, hemin-induced 56

PAGE 57

overexpression of HO-1 did not protect myotubes against H 2 O 2 -mediated oxidative stress (i.e., protection against lipid per oxidation). Therefore, these results differ from our in vivo results where overexpression of HO-1 protected the diaphragm against MV-induced oxidative damage. Several possibilities exist for these divergent findings. First, although H 2 O 2 is widely used to impose an oxidative stress-like en vironment in cell culture, using H 2 O 2 alone does not simulate the normal in vivo environment where numerous ra dicals and reactive oxygen species (i.e. superoxide, nitric oxide, hydroxyl radicals, etc) are continually produced within skeletal muscle (4). Hence, it is possible that HO-1 possesses antioxidant scavenging properties against reactive oxygen species other than H 2 O 2 Another explanation for our di vergent findings invol ves the complexity of determining both a physiologically releva nt concentration of H 2 O 2 and duration of exposure to provide an oxidative challenge in myotubes. Alt hough intracellular conc entrations of H 2 O 2 have been estimated to be < 1 m in cells not experiencing oxidative stress (18), previ ous investigators have exposed cells to a wide range of H 2 O 2 concentrations (25 m 1mM) (65, 80, 90, 96, 128),. This disparity creates difficulty in determining the most physiologic and re levant conditions for an oxidant challenge. In our in vitro experiments, we exposed myotubes to H 2 O 2 concentrations of 100 M and below because higher concentrations of H 2 O 2 diminish cellular viability (unpublished observations). Therefore, we reas oned that it was appropriate to expose myotubes to H 2 O 2 concentrations that would induce oxidative stress without decreasi ng cellular viability. Nonetheless, it is possible that ou r exposure of myotubes to exogenous H 2 O 2 resulted in intracellular levels of H 2 O 2 that surpass the levels observed in diaphragm fibers during prolonged MV. That is, it is feasible that the H 2 O 2 levels used in our experiments overwhelmed the antioxidant properties of HO-1 and the other endogenous antioxidants. Moreover, although H 2 O 2 57

PAGE 58

can pass freely into the cell, concentrat ions within cellular compartments in in vitro experiments do not necessarily reflect those found in in vivo conditions (18). Finally, the PO 2 of the environment (95% O 2 and PO 2 ~677 mmHg) commonly used during the growth and differentiation of myotubes is much higher than in vivo levels and may result in a myotube phenotype that differs markedly fr om skeletal muscle fibers deri ved from animals. Therefore, collectively, these concerns raise important que stions regarding the phys iological relevance of in vitro myotube models of oxidative stress. Conclusions and Future Directions This study provides the first in vivo evidence that overexpression of HO-1 functions as an antioxidant in the diaphragm during MV. Speci fically, these results demonstrate that HO-1 improves resistance to MV-induced oxidative stress. Furthermore, the induction of HO-1 in the diaphragm is effective in diminishing MV-i nduced protease (e.g., calpain and caspase-3) activation and is associated w ith a reduction in MV-i nduced diaphragmatic atrophy. Collectively, these are novel and important findings. Indeed, pr ior to the current experiments, the question of whether HO-1 functions as an antioxidant or pro-oxidant remained unanswered. Our results clearly indicate that overexpr ession of HO-1 provides protec tion against inactivity-induced oxidative stress in skeletal muscle fibers. Moreover, our findings suggest that pharmacological induction of HO-1 expression coul d be a potential therapeutic st rategy to retard MV-induced oxidative stress and atrophy in the diaphragm duri ng prolonged MV. This is clinically significant because MV-induced diaphragmatic weakness plays an important role in weaning difficulties following MV. Future studies should focus on elucidating the pathways responsible for oxidant production in the diaphragm during prolonged MV and on the mechanism(s) that are accountable for oxidative stress-induced activati on of both calpain and caspase3. A complete understanding of 58

PAGE 59

these two important issues is essential to develop effective and safe countermeasures for preventing respiratory muscle atrophy and weakness during prolonged MV. Development of a clinically useful countermeasure to prevent or retard MV-induced diaphragmatic atrophy and weakness would potentially maintain normal insp iratory muscle functi on during prolonged MV, which could permit patients to wean from the ventilator more successfully. 59

PAGE 60

LIST OF REFERENCES 1. Andreyev AY, Kushnareva YE, and Starkov AA. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70: 200-214, 2005. 2. Anzueto A, Peters JI, Tobin MJ, de los Santos R, Seidenfeld JJ, Moore G, Cox WJ, and Coalson JJ. Effects of prolonged controlled mech anical ventilation on diaphragmatic function in healthy adult baboons. Crit Care Med 25: 1187-1190, 1997. 3. Applegate LA, Luscher P, and Tyrrell RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res 51: 974-978, 1991. 4. Arbogast S and Reid MB. Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide. Am J Physiol Regul Integr Comp Physiol 287: R698-705, 2004. 5. Attaix D, Ventadour S, Codran A, Bechet D, Taillandier D, and Combaret L. The ubiquitin-proteasome system and skeletal muscle wasting. Essays Biochem 41: 173-186, 2005. 6. Baar K, Nader G, and Bodine S. Resistance exercise, muscle loading/unloading and the control of muscle mass. Essays Biochem 42: 61-74, 2006. 7. Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton JW, and Vercellotti GM. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem 267: 18148-18153, 1992. 8. Balla J, Jacob HS, Balla G, Nath K, Eaton JW, and Vercellotti GM. Endothelial-cell heme uptake from heme proteins: induction of sensitization and desensitization to oxidant damage. Proc Natl Acad Sci U S A 90: 9285-9289, 1993. 9. Balla J, Jacob HS, Balla G, Nath K, and Vercellotti GM. Endothelial cell heme oxygenase and ferritin induction by heme proteins : a possible mechanism limiting shock damage. Trans Assoc Am Physicians 105: 1-6, 1992. 10. Baranano DE, Rao M, Ferris CD, and Snyder SH. Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci U S A 99: 16093-16098, 2002. 11. Barreiro E, Comtois AS, Gea J, Laubach VE, and Hussain SN. Protein tyrosine nitration in the ventilatory muscles: role of nitric oxide synthases. Am J Respir Cell Mol Biol 26: 438-446, 2002. 12. Barreiro E, Comtois AS, Mohammed S, Lands LC, and Hussain SN. Role of heme oxygenases in sepsis-induced diaphragmatic c ontractile dysfunction and oxidative stress. Am J Physiol Lung Cell Mol Physiol 283: L476-484, 2002. 13. Bartoli M and Richard I. Calpains in muscle wasting. Int J Biochem Cell Biol 37: 21152133, 2005. 60

PAGE 61

14. Bernard N, Matecki S, Py G, Lo pez S, Mercier J, and Capdevila X. Effects of prolonged mechanical ventilation on respirator y muscle ultrastructure and mitochondrial respiration in rabbits. Intensive Care Med 29: 111-118, 2003. 15. Betters JL, Criswell DS, Shanely RA, Van Gammeren D, Falk D, Deruisseau KC, Deering M, Yimlamai T, and Powers SK. Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis. Am J Respir Crit Care Med 170: 1179-1184, 2004. 16. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, and Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704-1708, 2001. 17. Cadenas E and Davies KJ. Mitochondrial free radical ge neration, oxidative stress, and aging. Free Radic Biol Med 29: 222-230, 2000. 18. Chance B, Sies H, and Boveris A. Hydroperoxide metabolis m in mammalian organs. Physiol Rev 59: 527-605, 1979. 19. Clark JE, Foresti R, Gr een CJ, and Motterlini R. Dynamics of haem oxygenase-1 expression and bilirubin production in cellu lar protection against oxidative stress. Biochem J 348 Pt 3: 615-619, 2000. 20. Communal C, Sumandea M, de Tombe P, Narula J, Solaro RJ, and Hajjar RJ. Functional consequences of caspase activation in cardiac myocytes. Proc Natl Acad Sci U S A 99: 6252-6256, 2002. 21. Criswell DS, Shanely RA, Betters JJ, McKenzie MJ, Sellman JE, Van Gammeren DL, and Powers SK. Cumulative effects of aging and m echanical ventilation on in vitro diaphragm function. Chest 124: 2302-2308, 2003. 22. DeRuisseau KC, Kavazis AN, Deering MA Falk DJ, Van Gammeren D, Yimlamai T, Ordway GA, and Powers SK. Mechanical ventilation induces alterations of the ubiquitinproteasome pathway in the diaphragm. J Appl Physiol 98: 1314-1321, 2005. 23. Deruisseau KC, Kavazis AN, and Powers SK. Selective downregul ation of ubiquitin conjugation cascade mRNA occurs in the senescent rat soleus muscle. Exp Gerontol 40: 526-531, 2005. 24. Duan X, Berthiaume F, Yarmush D, and Yarmush ML. Proteomic analysis of altered protein expression in skel etal muscle of rats in a hypermet abolic state induced by burn sepsis. Biochem J 397: 149-158, 2006. 25. Erdmann K, Grosser N, Schipporeit K, and Schroder H. The ACE inhibitory dipeptide Met-Tyr diminishes free radical forma tion in human endothelial cells via induction of heme oxygenase-1 and ferritin. J Nutr 136: 2148-2152, 2006. 61

PAGE 62

26. Erdmann K, Grosser N, and Schroder H. L-methionine reduces oxidant stress in endothelial cells: role of heme oxyge nase-1, ferritin, and nitric oxide. Aaps J 7: E195-200, 2005. 27. Esteban A, Alia I, Ibanez J, Benito S, and Tobin MJ. Modes of mechanical ventilation and weaning. A national survey of Spanish hos pitals. The Spanish Lung Failure Collaborative Group. Chest 106: 1188-1193, 1994. 28. Falk DJ, Deruisseau KC, Van Gammere n DL, Deering MA, Kavazis AN, and Powers SK. Mechanical ventilation promotes redox status alterations in the diaphragm. J Appl Physiol 101: 1017-1024, 2006. 29. Fantini GA and Yoshioka T. Deferoxamine prevents lipi d peroxidation and attenuates reoxygenation injury in postischemic skeletal muscle. Am J Physiol 264: H1953-1959, 1993. 30. Fredriksson K, Radell P, Eriksson LI, Hultenby K, and Rooyackers O. Effect of prolonged mechanical ventilation on diaphr agm muscle mitochondria in piglets. Acta Anaesthesiol Scand 49: 1101-1107, 2005. 31. Gayan-Ramirez G, de Paepe K, Cadot P, and Decramer M. Detrimental effects of short-term mechanical ventilation on di aphragm function and IGF-I mRNA in rats. Intensive Care Med 29: 825-833, 2003. 32. Gayan-Ramirez G and Decramer M. Effects of mechanical ventilation on diaphragm function and biology. Eur Respir J 20: 1579-1586, 2002. 33. Gomes-Marcondes MC and Tisdale MJ. Induction of protein catabolism and the ubiquitin-proteasome pathway by mild oxidative stress. Cancer Lett 180: 69-74, 2002. 34. Gonzales S, Erario MA, and Tomaro ML. Heme oxygenase-1 induction and dependent increase in ferritin. A protective antioxidant stratagem in hemin-treated rat brain. Dev Neurosci 24: 161-168, 2002. 35. Halliwell B and Gutteridge J. Free Radicals in Biology and Medicine London: Oxford Press, 1999. 36. Hayes JD and McLellan LI. Glutathione and glutathione-d ependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 31: 273-300, 1999. 37. Hazan I, Dana R, Granot Y, and Levy R. Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogenactivated protein kinase, cytosolic phospholipase A2 and NADPH oxidase. Biochem J 326 ( Pt 3): 867-876, 1997. 38. Hellsten Y. The role of xanthine oxidase in exercise. In: Handbook of Oxidants and Antioxidants in Exercise edited by al. CSe. Amsterdam: Elsevier Press, 2000, p. 153-176. 62

PAGE 63

39. Hill NS and Levy MM. Ventilator Management Strategies for Critical Care New York, NY 10016: Marcel Dekker, Inc., 2001. 40. Hool LC and Corry B. Redox control of calcium channels: from mechanisms to therapeutic opportunities. Antioxid Redox Signal 9: 409-435, 2007. 41. Hunter RB, Mitchell-Felton H, Essig DA, and Kandarian SC. Expression of endoplasmic reticulum stress proteins during skeletal muscle disuse atrophy. Am J Physiol Cell Physiol 281: C1285-1290, 2001. 42. Jaber S, Sebbane M, Koechlin C, Hayot M, Capdevila X, Eledjam JJ, Prefaut C, Ramonatxo M, and Matecki S. Effects of short vs. prol onged mechanical ventilation on antioxidant systems in piglet diaphragm. Intensive Care Med 31: 1427-1433, 2005. 43. Javesghani D, Magder SA, Barreiro E, Quinn MT, and Hussain SN. Molecular characterization of a superoxi de-generating NAD(P)H oxidase in the ventilatory muscles. Am J Respir Crit Care Med 165: 412-418, 2002. 44. Kaminski HJ and Andrade FH. Nitric oxide: biologic eff ects on muscle and role in muscle diseases. Neuromuscul Disord 11: 517-524, 2001. 45. Kandarian SC and Stevenson EJ. Molecular events in skel etal muscle during disuse atrophy. Exerc Sport Sci Rev 30: 111-116, 2002. 46. Keyse SM, Applegate LA, Tromvoukis Y, and Tyrrell RM. Oxidant stress leads to transcriptional activation of the human heme oxy genase gene in cultur ed skin fibroblasts. Mol Cell Biol 10: 4967-4969, 1990. 47. Keyse SM and Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiati on, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci U S A 86: 99-103, 1989. 48. Keyse SM and Tyrrell RM. Induction of the heme oxygenase gene in human skin fibroblasts by hydrogen peroxide and UVA (365 nm) radiation: evidence for the involvement of the hydroxyl radical. Carcinogenesis 11: 787-791, 1990. 49. Kim YC, Masutani H, Yamaguchi Y, Itoh K, Yamamoto M, and Yodoi J. Hemininduced activation of the thioredoxin gene by Nrf 2. A differential regulation of the antioxidant responsive element by a switc h of its binding factors. J Biol Chem 276: 18399-18406, 2001. 50. Knisely AS, Leal SM, and Singer DB. Abnormalities of diaphragmatic muscle in neonates with ventilated lungs. J Pediatr 113: 1074-1077, 1988. 51. Kondo H, Miura M, and Itokawa Y. Antioxidant enzyme systems in skeletal muscle atrophied by immobilization. Pflugers Arch 422: 404-406, 1993. 63

PAGE 64

52. Kondo H, Miura M, and Itokawa Y. Oxidative stress in skel etal muscle atrophied by immobilization. Acta Physiol Scand 142: 527-528, 1991. 53. Kondo H, Miura M, Kodama J, Ahmed SM, and Itokawa Y. Role of iron in oxidative stress in skeletal muscle atrophied by immobilization. Pflugers Arch 421: 295-297, 1992. 54. Kondo H, Miura M, Nakagaki I, Sasaki S, and Itokawa Y. Trace element movement and oxidative stress in skeletal muscle atrophied by immobilization. Am J Physiol 262: E583-590, 1992. 55. Kondo H, Nakagaki I, Sasaki S, Hori S, and Itokawa Y. Mechanism of oxidative stress in skeletal muscle atrophied by immobilization. Am J Physiol 265: E839-844, 1993. 56. Ku Z, Yang J, Menon V, and Thomason DB. Decreased polysomal HSP-70 may slow polypeptide elongation during skeletal muscle atrophy. Am J Physiol 268: C1369-1374, 1995. 57. Kvam E, Hejmadi V, Ryter S, Pourzand C, and Tyrrell RM. Heme oxygenase activity causes transien t hypersensitivity to oxid ative ultraviolet A radi ation that depends on release of iron from heme. Free Radic Biol Med 28: 1191-1196, 2000. 58. Lamb NJ, Quinlan GJ, Mumby S, Evans TW, and Gutteridge JM. Haem oxygenase shows pro-oxidant activity in microsomal and cellu lar systems: implications for the release of low-molecular-mass iron. Biochem J 344 Pt 1: 153-158, 1999. 59. Lawler JM and Powers SK. Oxidative stress, antioxidant status, and the contracting diaphragm. Can J Appl Physiol 23: 23-55, 1998. 60. Le Bourdelles G, Viires N, Boczkowski J, Seta N, Pavlovic D, and Aubier M. Effects of mechanical ventilation on diaphragmatic contract ile properties in rats. Am J Respir Crit Care Med 149: 1539-1544, 1994. 61. Lemiare F. Difficult weaning. Intensive Care Med 19: S69-S73, 1993. 62. Levine S. TN, M. Friscia, L.R. Kaiser, J.B. Shrager. Ventilator-Induced Atrophy in Human Diaphragm Myofibers. Proc American Thoracic Society (International Conference) : Abstract p:A27, 2006. 63. Li YP, Atkins CM, Sweatt JD, and Reid MB. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes. Antioxid Redox Signal 1: 97-104, 1999. 64. Li YP, Chen Y, John J, Moylan J, Jin B, Mann DL, and Reid MB. TNF-alpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle. Faseb J 19: 362-370, 2005. 64

PAGE 65

65. Li YP, Chen Y, Li AS, and Reid MB. Hydrogen peroxide stimulates ubiquitinconjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes. Am J Physiol Cell Physiol 285: C806-812, 2003. 66. Li YP and Reid MB. NF-kappaB mediates the protei n loss induced by TNF-alpha in differentiated skeletal muscle myotubes. Am J Physiol Regul Integr Comp Physiol 279: R11651170, 2000. 67. Li YP, Schwartz RJ, Waddell ID, Holloway BR, and Reid MB. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-k appaB activation in response to tumor necrosis factor alpha. Faseb J 12: 871-880, 1998. 68. Lin F and Girotti AW. Hemin-enhanced resistance of human leukemia cells to oxidative killing: antisense determination of ferritin involvement. Arch Biochem Biophys 352: 51-58, 1998. 69. Maes K, Testelmans D, Powers S, Decramer M, and Gayan-Ramirez G. Leupeptin inhibits ventilator-induced diaphragm dysfunction in rats. Am J Respir Crit Care Med 175: 11341138, 2007. 70. McClung JM, A.N. Kavazis, M.A. Whidde n, K.C. DeRuisseau, D.J. Falk, D.S. Criswell, S.K. Powers. Trolox attenuates ventilation musc le atrophy independent of protein kinase B (PKB/Akt) signaling. Am J Respir Crit Care Med in review, 2007. 71. McClung JM, Kavazis AN, DeRuisseau KC, Falk DJ, Deering MA, Lee Y, Sugiura T, and Powers SK. Caspase-3 regulation of diaphragm myonuclear domain during mechanical ventilation-induced atrophy. Am J Respir Crit Care Med 175: 150-159, 2007. 72. McClung JM, Kavazis AN, Whidden MA, Deruisseau KC, Falk DJ, Criswell DS, and Powers SK. Antioxidant Administration Attenuate s Mechanical Ventilation-Induced Rat Diaphragm Muscle Atrophy Independent of Protein Kinase B (PKB/Akt) Signaling. J Physiol, 2007. 73. McLoughlin TJ, Tsivitse SK, Edwards JA, Aiken BA, and Pizza FX. Deferoxamine reduces and nitric oxide syntha se inhibition increases neutro phil-mediated myotube injury. Cell Tissue Res 313: 313-319, 2003. 74. Metzger JM, Scheidt KB, and Fitts RH. Histochemical and physiological characteristics of the rat diaphragm. J Appl Physiol 58: 1085-1091, 1985. 75. Mizuno M. Human respiratory muscles: fi bre morphology and capillary supply. Eur Respir J 4: 587-601, 1991. 76. Motterlini R, Foresti R, Intaglietta M, and Winslow RM. NO-mediated activation of heme oxygenase: endogenous cytoprotection ag ainst oxidative stress to endothelium. Am J Physiol 270: H107-114, 1996. 65

PAGE 66

77. Nath KA, Balla G, Vercellotti GM, Balla J, Jacob HS, Levitt MD, and Rosenberg ME. Induction of heme oxygenase is a rapid, protect ive response in rhabdom yolysis in the rat. J Clin Invest 90: 267-270, 1992. 78. Nguyen HX and Tidball JG. Expression of a muscle-speci fic, nitric oxide synthase transgene prevents muscle membrane injury and reduces muscle inflammation during modified muscle use in mice. J Physiol 550: 347-356, 2003. 79. Nin N, Cassina A, Boggia J, Alfonso E, Botti H, Peluffo G, Trostchansky A, Batthyany C, Radi R, Rubbo H, and Hurtado FJ. Septic diaphragmatic dysfunction is prevented by Mn(III)porphyrin therapy and indu cible nitric oxide s ynthase inhibition. Intensive Care Med 30: 2271-2278, 2004. 80. Orzechowski A, Grizard J, Jank M, Ga jkowska B, Lokociejewska M, ZaronTeperek M, and Godlewski M. Dexamethasone-mediated regulation of death and differentiation of muscle cel ls. Is hydrogen peroxide involved in the process? Reprod Nutr Dev 42: 197-216, 2002. 81. Pavlovic D and Wendt M. Diaphragm pacing during prolon ged mechanical ventilation of the lungs could prevent from respiratory muscle fatigue. Med Hypotheses 60: 398-403, 2003. 82. Poole DC, Sexton WL, Farkas GA, Powers SK, and Reid MB. Diaphragm structure and function in health and disease. Med Sci Sports Exerc 29: 738-754, 1997. 83. Powers SK, Demirel HA, Coombes JS, Fl etcher L, Calliaud C, Vrabas I, and Prezant D. Myosin phenotype and bioenergetic charac teristics of rat respiratory muscles. Med Sci Sports Exerc 29: 1573-1579, 1997. 84. Powers SK, Kavazis AN, and DeRuisseau KC. Mechanisms of disuse muscle atrophy: role of oxidative stress. Am J Physiol Regul Integr Comp Physiol 288: R337-344, 2005. 85. Powers SK, Kavazis AN, and McClung JM. Oxidative stress and disuse muscle atrophy. J Appl Physiol 2007. 86. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M, Van Gammeren D, Cicale M, and Dodd SL. Mechanical ventilation result s in progressive contractile dysfunction in the diaphragm. J Appl Physiol 92: 1851-1858, 2002. 87. Racz GZ, Gayan-Ramirez G, Testelmans D, Cadot P, De Paepe K, Zador E, Wuytack F, and Decramer M. Early changes in rat diaphr agm biology with mechanical ventilation. Am J Respir Crit Care Med 168: 297-304, 2003. 88. Radell PJ, Remahl S, Nichols DG, and Eriksson LI. Effects of prolonged mechanical ventilation and inactivity on piglet diaphragm function. Intensive Care Med 28: 358-364, 2002. 66

PAGE 67

89. Rando TA. Oxidative stress and the pathoge nesis of muscular dystrophies. Am J Phys Med Rehabil 81: S175-186, 2002. 90. Rohrbach S, Gruenler S, Teschner M, and Holtz J. The thioredoxin system in aging muscle: key role of mitochondrial thioredoxin re ductase in the protective effects of caloric restriction? Am J Physiol Regul Integr Comp Physiol 291: R927-935, 2006. 91. Ryter SW, Alam J, and Choi AM. Heme oxygenase-1/carb on monoxide: from basic science to therapeutic applications. Physiol Rev 86: 583-650, 2006. 92. Ryter SW and Tyrrell RM. The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both proand antioxidant properties. Free Radic Biol Med 28: 289-309, 2000. 93. Sassoon CS. Ventilator-associated diaphragmatic dysfunction. Am J Respir Crit Care Med 166: 1017-1018, 2002. 94. Sassoon CS, Caiozzo VJ, Manka A, and Sieck GC. Altered diaphragm contractile properties with controlled mechanical ventilation. J Appl Physiol 92: 2585-2595, 2002. 95. Sassoon CS, Zhu E, and Caiozzo VJ. Assist-control mechanical ventilation attenuates ventilator-induced diaphragmatic dysfunction. Am J Respir Crit Care Med 170: 626-632, 2004. 96. Savini I, Rossi A, Catani MV, Ceci R, and Avigliano L. Redox regulation of vitamin C transporter SVCT2 in C2C12 myotubes. Biochem Biophys Res Commun 361: 385-390, 2007. 97. Servais S, Letexier D, Favier R, Duchamp C, and Desplanches D. Prevention of unloading-induced atrophy by vitamin E supplem entation: links between oxidative stress and soleus muscle proteolysis? Free Radic Biol Med 42: 627-635, 2007. 98. Shanely RA, Coombes JS, Zergerogl u AM, Webb AI, and Powers SK. Short-duration mechanical ventilation enhances diaphragmatic fatigue resistance but impairs force production. Chest 123: 195-201, 2003. 99. Shanely RA, Van Gammeren D, Deruisseau KC, Zergeroglu AM, McKenzie MJ, Yarasheski KE, and Powers SK. Mechanical ventilation depresse s protein synthesis in the rat diaphragm. Am J Respir Crit Care Med 170: 994-999, 2004. 100. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T, Enns D, Belcastro A, and Powers SK. Mechanical ventilation-induc ed diaphragmatic atrophy is associated with oxidative injury and increased proteolytic activity. Am J Respir Crit Care Med 166: 1369-1374, 2002. 101. Siu PM and Alway SE. Mitochondria-associated apoptotic signalling in denervated rat skeletal muscle. J Physiol 565: 309-323, 2005. 67

PAGE 68

102. Stamler JS and Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol Rev 81: 209-237, 2001. 103. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, and Ames BN. Bilirubin is an antioxidant of possible p hysiological importance. Science 235: 1043-1046, 1987. 104. Stofan DA, Callahan LA, Di MA Nethery DE, and Supinski GS. Modulation of release of reactive oxyge n species by the contracting diaphragm. Am J Respir Crit Care Med 161: 891-898, 2000. 105. Sultan KR, Henkel B, Terl ou M, and Haagsman HP. Quantification of hormoneinduced atrophy of large myotubes from C2C 12 and L6 cells: atrophy-inducible and atrophyresistant C2C12 myotubes. Am J Physiol Cell Physiol 290: C650-659, 2006. 106. Supinski G, Nethery D, Stofan D, Hirschfield W, and DiMarco A. Diaphragmatic lipid peroxidation in chronically loaded rats. J Appl Physiol 86: 651-658, 1999. 107. Supinski G, Nethery D, Stofan D, Szweda L, and DiMarco A. Oxypurinol administration fails to prevent free radical-med iated lipid peroxidation during loaded breathing. J Appl Physiol 87: 1123-1131, 1999. 108. Supinski G, Stofan D, Callahan LA, Nethery D, Nosek TM, and DiMarco A. Peroxynitrite induces contract ile dysfunction and lipid per oxidation in the diaphragm. J Appl Physiol 87: 783-791, 1999. 109. Supinski GS and Callahan LA. Hemin prevents cardiac an d diaphragm mitochondrial dysfunction in sepsis. Free Radic Biol Med 40: 127-137, 2006. 110. Suttner DM and Dennery PA. Reversal of HO-1 related cy toprotection with increased expression is due to reactive iron. Faseb J 13: 1800-1809, 1999. 111. Taillandier D, Aurousseau E, Meynial-Denis D, Bechet D, Ferrara M, Cottin P, Ducastaing A, Bigard X, Guezennec CY, Schmid HP, and et al. Coordinate activation of lysosomal, Ca 2+-activated and ATP-ubiquitindependent proteinases in the unweighted rat soleus muscle. Biochem J 316 ( Pt 1): 65-72, 1996. 112. Taille C, Foresti R, Lanone S, Zedda C, Green C, Aubier M, Motterlini R, and Boczkowski J. Protective role of heme oxygenases against endotoxin-induced diaphragmatic dysfunction in rats. Am J Respir Crit Care Med 163: 753-761, 2001. 113. Tenhunen R, Marver HS, and Schmid R. The enzymatic catabolism of hemoglobin: stimulation of microsomal heme oxygenase by hemin. J Lab Clin Med 75: 410-421, 1970. 114. Tenhunen R, Marver HS, and Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci U S A 61: 748-755, 1968. 68

PAGE 69

115. Tenhunen R, Marver HS, and Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J Biol Chem 244: 6388-6394, 1969. 116. Tidball JG and Spencer MJ. Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isof orm expression during murine muscle disuse. J Physiol 545: 819-828, 2002. 117. Van Bergen P, Rauhala P, Spooner CM, and Chiueh CC. Hemoglobin and ironevoked oxidative stress in the brain: protection by bile pigments, manganese and Snitrosoglutathione. Free Radic Res 31: 631-640, 1999. 118. Van Gammeren D, Falk DJ, Deering MA Deruisseau KC, and Powers SK. Diaphragmatic Nitric Oxide Synthase is Not Induced During Mechanical Ventilation. J Appl Physiol 2006. 119. Van Gammeren D, Falk DJ, DeRuisseau KC Sellman JE, Decramer M, and Powers SK. Reloading the diaphragm following mechani cal ventilation does not promote injury. Chest 127: 2204-2210, 2005. 120. Vassilakopoulos T. Ventilator-induced diaphragm dysfunction: the clinical relevance of animal models. Intensive Care Med 2007. 121. Ventadour S and Attaix D. Mechanisms of skeletal muscle atrophy. Curr Opin Rheumatol 18: 631-635, 2006. 122. Vile GF, Basu-Modak S, Waltner C, and Tyrrell RM. Heme oxygenase 1 mediates an adaptive response to oxidative stre ss in human skin fibroblasts. Proc Natl Acad Sci U S A 91: 2607-2610, 1994. 123. Wasserman WW and Fahl WE. Functional antioxidant responsive elements. Proc Natl Acad Sci U S A 94: 5361-5366, 1997. 124. Watson AC, Hughes PD, Louise Harris M, Hart N, Ware RJ, Wendon J, Green M, and Moxham J. Measurement of twitch transdiaphrag matic, esophageal, and endotracheal tube pressure with bilateral anterolateral magnetic phrenic nerve stimulation in patients in the intensive care unit. Crit Care Med 29: 1325-1331, 2001. 125. Whidden MAF, D.J.; Smuder, A.J.; McClung, J.M.; and S.K. Powers. Oxypurinol attenuates mechanical ventilation-induced di aphragmatic oxidative stress and contractile dysfunction. Faseb J 21, 2007. 126. Wu TW, Wu J, Li RK, Mickle D, and Carey D. Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage. Biochem Cell Biol 69: 683-688, 1991. 69

PAGE 70

127. Yang L, Luo J, Bourdon J, Lin MC, Gottfried SB, and Petrof BJ. Controlled mechanical ventilation leads to remodeling of the rat diaphragm. Am J Respir Crit Care Med 166: 1135-1140, 2002. 128. Yoshikawa A, Saito Y, and Maruyama K. Lignan compounds and 4,4'dihydroxybiphenyl protect C2C12 cells agai nst damage from oxidative stress. Biochem Biophys Res Commun 344: 394-399, 2006. 129. Zergeroglu MA, McKenzie MJ, Shanely RA, Van Gammeren D, DeRuisseau KC, and Powers SK. Mechanical ventilation-induced oxidative stress in the diaphragm. J Appl Physiol 95: 1116-1124, 2003. 130. Zhang P, Chen X, and Fan M. Signaling mechanisms involved in disuse muscle atrophy. Med Hypotheses 2007. 131. Zhu E, Sassoon CS, Nelson R, Pham HT, Zhu L, Baker MJ, and Caiozzo VJ. Early effects of mechanical ventilation on isotonic contract ile properties and MAF-box gene expression in the diaphragm. J Appl Physiol 99: 747-756, 2005. 70

PAGE 71

BIOGRAPHICAL SKETCH Darin J. Falk was born in Columbus, Nebraska. He attained a Bachelor of Science degree from the University of Nebras ka-Kearney. Following graduation, he pursued a Masters degree in exercise physiology and graduated from Univ ersity of Nebraska-Kearney in 2004. Deciding to focus his career in basic scien ce, Darin began his doctoral work at the University of Florida in 2002 under the direction of Scott K. Powers. Da rin focused his studies on the mechanisms underlying diaphragmatic atrophy and dysfunction induced by m echanical ventilation. He received his PhD in 2007. 71