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Doxorubicin-Induced Cardiac and Skeletal Myopathy

Permanent Link: http://ufdc.ufl.edu/UFE0044927/00001

Material Information

Title: Doxorubicin-Induced Cardiac and Skeletal Myopathy the Role of Mitochondrial Reactive Oxygen Species Emission and Calpain Activation
Physical Description: 1 online resource (96 p.)
Language: english
Creator: Min, Kisuk
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: calpain -- doxorubicin -- mitochondria -- ros
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: Doxorubicin (DOX) is an antitumor agent used to treat a variety of cancers. Although DOX is a highly effective anti-tumor treatment, DOX has been shown to induce detrimental effects in several tissues, including cardiac and skeletal muscle. At present, a complete understanding of the mechanisms responsible for DOX-mediated cellular toxicity remains elusive. Although the exact causal mechanism of DOX-mediated muscle myopathy is unclear, it appears that an increased cellular production of reactive oxygen species (ROS) is a contributing factor. Specifically, it has been demonstrated that DOX-induced ROS are generated by NAD(P)H oxidoreductases, such as  mitochondrial NADH dehydrogenase. Nonetheless, it remains unknown if mitochondrial ROS production is a requirement for DOX-induced cardiac and skeletal muscle myopathy. Furthermore, treatment of animals with DOX activates the cysteine protease calpain in both cardiac and skeletal muscle fibers. This is significant because calpain activation can promote cellular injury. Nonetheless, it is unknown if calpain activation is a requirement for DOX-induced cardiac and skeletal muscle myopathy. Therefore, this projection tested the central hypothesis that mitochondrial ROS production plays a required role in DOX-induced cardiac and skeletal muscle myopathy and that ROS-mediated activation of calpain is a key contributor to DOX-induced cardiac and skeletal muscle myopathy. Our study reveals that mitochondria are the dominant source of oxidant production in the cardiac and skeletal muscle during DOX administration. Indeed, mitochondrial ROS emission is a required upstream signal to activate key proteases during DOX administration. Furthermore, our data also reveals that calpain activation is a requirement for DOX-induced myopathy in the heart and skeletal muscles, and calpain inhibition prevents DOX-induced damage to cardiac and skeletal muscles. Collectively, the current findings suggest that mitochondrial ROS emission and calpain activation contribute to the development of DOX-induced myopathy in cardiac and skeletal muscles.
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 Kisuk Min.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044927:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044927/00001

Material Information

Title: Doxorubicin-Induced Cardiac and Skeletal Myopathy the Role of Mitochondrial Reactive Oxygen Species Emission and Calpain Activation
Physical Description: 1 online resource (96 p.)
Language: english
Creator: Min, Kisuk
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: calpain -- doxorubicin -- mitochondria -- ros
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: Doxorubicin (DOX) is an antitumor agent used to treat a variety of cancers. Although DOX is a highly effective anti-tumor treatment, DOX has been shown to induce detrimental effects in several tissues, including cardiac and skeletal muscle. At present, a complete understanding of the mechanisms responsible for DOX-mediated cellular toxicity remains elusive. Although the exact causal mechanism of DOX-mediated muscle myopathy is unclear, it appears that an increased cellular production of reactive oxygen species (ROS) is a contributing factor. Specifically, it has been demonstrated that DOX-induced ROS are generated by NAD(P)H oxidoreductases, such as  mitochondrial NADH dehydrogenase. Nonetheless, it remains unknown if mitochondrial ROS production is a requirement for DOX-induced cardiac and skeletal muscle myopathy. Furthermore, treatment of animals with DOX activates the cysteine protease calpain in both cardiac and skeletal muscle fibers. This is significant because calpain activation can promote cellular injury. Nonetheless, it is unknown if calpain activation is a requirement for DOX-induced cardiac and skeletal muscle myopathy. Therefore, this projection tested the central hypothesis that mitochondrial ROS production plays a required role in DOX-induced cardiac and skeletal muscle myopathy and that ROS-mediated activation of calpain is a key contributor to DOX-induced cardiac and skeletal muscle myopathy. Our study reveals that mitochondria are the dominant source of oxidant production in the cardiac and skeletal muscle during DOX administration. Indeed, mitochondrial ROS emission is a required upstream signal to activate key proteases during DOX administration. Furthermore, our data also reveals that calpain activation is a requirement for DOX-induced myopathy in the heart and skeletal muscles, and calpain inhibition prevents DOX-induced damage to cardiac and skeletal muscles. Collectively, the current findings suggest that mitochondrial ROS emission and calpain activation contribute to the development of DOX-induced myopathy in cardiac and skeletal muscles.
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 Kisuk Min.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Powers, Scott K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044927:00001


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1 DOXORUBICIN INDUCED CARDIAC AND SKELETAL MYOPATHY: THE ROLE OF M ITOCHONDRIAL REACTIVE OXYGEN SP EC I ES EMISSION AND CALPAIN ACTIVATION By KISUK MIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Kisuk Min

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3 To my family and friends for their constant support and to the people who have played a significant role in my education

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4 ACKNOWLEDGMENTS First, I would like to sincerely thank my mentor Dr Scott Powers for his guidance, understanding and continuous suppor t during my graduate education. Also, I commend iswell and Dr. Peter Adhihetty for their direction and support th roughout my graduate studies. I also wish to thank all the laboratory members who played a role in my achievements. Further, I am thankful for my wife an d my two lovely children for their fai th and consideration. Lastly, I am extremely grateful to my parents for their encouragement and prayer s for me.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURE S ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 2 LITERATURE REVIEW ................................ ................................ .......................... 16 Mechanisms for DOX induced Oxidative Stress ................................ ..................... 17 Mechanisms for DOX induced Cardiac and Skeletal Muscle Damage and Contractile Dysfunction ................................ ................................ ........................ 17 Calpains ................................ ................................ ................................ ........... 18 Caspase 3 ................................ ................................ ................................ ........ 18 Ubiquitin Proteasome System ................................ ................................ .......... 18 Autophagic/Lysosomal Proteases ................................ ................................ .... 19 Summary ................................ ................................ ................................ ................ 20 3 MATERIALS AND METHODS ................................ ................................ ................ 22 Anim als ................................ ................................ ................................ ................... 22 Animal Model Justification ................................ ................................ ................ 22 Animal Housing and Diet ................................ ................................ .................. 22 Experiment 1 ................................ ................................ ................................ ........... 23 Experimental Des ign ................................ ................................ ........................ 23 Experimental Protocol ................................ ................................ ...................... 23 Statistical Analysis ................................ ................................ ............................ 24 Experiment 2 ................................ ................................ ................................ ........... 24 Experimental Design ................................ ................................ ........................ 24 Experime ntal Protocol ................................ ................................ ...................... 24 Statistical Analysis ................................ ................................ ............................ 25 General Methods ................................ ................................ ................................ .... 26 Functional Measures ................................ ................................ ........................ 26 Histolog ical Measures ................................ ................................ ...................... 27 Biochemical Measures ................................ ................................ ..................... 28 Mitochondrial Measures ................................ ................................ ................... 29

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6 4 RESULTS ................................ ................................ ................................ ............... 33 Experiment 1: Systemic Response to Doxorubicin Administration .......................... 33 Cardiac Function ................................ ................................ .............................. 33 Physiological response to doxorubicin administration ................................ 33 Cardiac geometry and function ................................ ................................ .. 33 Diaphragm Contractile Function ................................ ................................ ....... 34 Skeletal Muscle Structure ................................ ................................ ................. 35 Mitochondrial Function ................................ ................................ ..................... 35 Redox Balance ................................ ................................ ................................ 35 Mitochondrial reactive oxygen species emission ................................ ....... 35 4 HNE ................................ ................................ ................................ ........ 36 Proteolytic Activity ................................ ................................ ............................ 36 Calpain ................................ ................................ ................................ ....... 36 Caspase 3 ................................ ................................ ................................ .. 37 Ubiquitin proteasome system ................................ ................................ ..... 37 Experiment 2: Systemic Response to Doxorubicin Administration .......................... 37 Cardiac Function ................................ ................................ .............................. 38 Physiological response to doxorubicin administration ................................ 38 Cardiac geometry and function ................................ ................................ .. 38 Diaphragm Contractile Function ................................ ................................ ....... 39 Skeletal Muscle Structure ................................ ................................ ................. 40 Mitochondrial Function ................................ ................................ ..................... 40 Redox Balance ................................ ................................ ................................ 40 Proteolytic Activity ................................ ................................ ............................ 41 Calp ain ................................ ................................ ................................ ....... 41 Caspase 3 ................................ ................................ ................................ .. 41 Ubiquitin proteasome system ................................ ................................ ..... 41 Lysosomal Protease ................................ ................................ ......................... 42 5 DISCUSSION ................................ ................................ ................................ ......... 81 Overview of Principal Findings ................................ ................................ ................ 81 DOX Administration Results in Cardiac and Skeletal Muscle Dysfunction .............. 81 Mitochondria are a Key Source of DOX Induced ROS Emission ............................ 82 Mitochondrial ROS Emission Promotes Protease Activation and Proteolysis Following DOX Administration ................................ ................................ ............. 83 Calpain Activation Leads to Muscle Dysfunction During DOX Administration ......... 85 Calpain Activity Contributes to DOX Induced Proteo lysis ................................ ....... 85 Calpain Activation Upregulates Lysosomal Proteases ................................ ............ 86 Conclusions and Future Directions ................................ ................................ ......... 87 LIST OF REFERENCES ................................ ................................ ............................... 89 BIOGRAPHIC AL SKETCH ................................ ................................ ............................ 96

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7 LIST OF TABLES Table page 4 1 Body weight changes for experiment 1. ................................ .............................. 43 4 2 Echocardiographic variables for experiment 1. ................................ ................... 43 4 3 Echocardiographic parameters for experiment 1. ................................ ............... 43 4 4 Body weight changes for the experiment 2 ................................ ......................... 43 4 5 Echocardiographic variables for experiment 2 ................................ .................... 44 4 6 Echocardiographic parameters for experiment 2 ................................ ................ 44

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8 LIST OF FIGURES Figure page 2 1 Proposed mechanisms of R OS formation by Doxorubicin ................................ 21 3 1 Experimental animal design used to determine if increased mitochondrial ROS production is a requirement for DOX induced damage and contractile dysfu nction in both cardiac and skeletal muscle. ................................ ................ 31 3 2 Experimental animal design used to determine if calpain activation is a requir ement for DOX induced damage to both cardiac and skeletal muscle. ..... 32 4 1 Body weight changes for experiment 1. ................................ .............................. 45 4 2 Representative M mode echocardiograms for experiment 1.. ............................ 46 4 3 Fractional shortening for experiment 1.. ................................ ............................. 46 4 4 Representative Doppler echocardiograms for experiment 1.. ............................. 47 4 5 Myocardial Performance Index for experiment 1. ................................ ............... 47 4 6 Diaphragm force frequency response ( in vitro ) of diaphragm samples for experiment 1.. ................................ ................................ ................................ ..... 48 4 7 Skeletal muscle cross sectional area for experiment 1.. ................................ ..... 49 4 8 Representative fluorescent staining of myosin heavy chain for experiment 1. ... 50 4 9 Mitochondrial respiratory function in permeabilized fiber from Heart muscle for experiment 1. ................................ ................................ ................................ 51 4 10 Mitochondrial respiratory function in permeabilized fiber from d i aphragm muscle for experiment 1 ................................ ................................ .................... 52 4 11 Rates of State 3 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fiber s for experiment 1. ................................ ................... 53 4 12 Rates of State 4 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experiment 1. ................................ ................... 54 4 13 The levels of 4 hydroxynonenal (4 HNE) were analyzed as an indic ator of lipid peroxidation via W estern blot ting for experiment 1. ................................ .... 55 4 14 Calpain activation in cardiac and skeletal muscle was determined via Western blotting for experimen t 1.. ................................ ................................ ..... 56

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9 4 15 Calpain specific spectrin breakdown product (SBDP) was determined for experiment 1.. ................................ ................................ ................................ ..... 57 4 16 Caspase 3 activation in the diaphragm was determined via Western blotting for experiment 1.. ................................ ................................ ................................ 58 4 17 Caspase 3 specific spectrin breakdown product was determined (SBDP) for experiment 1.. ................................ ................................ ................................ ..... 59 4 18 Apoptosis was determined by the terminal doxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for experiment 1. ................................ ........... 60 4 19 Representative images of sections statined using TUNEL assay for experiment 1. ................................ ................................ ................................ ...... 61 4 20 Atrogin 1 was determined via Western blotting for experiment 1. ....................... 62 4 21 MuRF 1 was determined via Western blotting for experiment 1. ........................ 63 4 22 Body weight changes for experiment 2 ................................ .............................. 64 4 23 Representative M mode echocardiograms for experiment 2. ............................. 65 4 24 Fractional shortening for experiment 2. ................................ .............................. 65 4 25 Representativ e Doppler echocardiograms for experiment 2. .............................. 66 4 26 Myocardial Performance Index for experiment 2. ................................ ............... 66 4 27 Diaphragm force frequency response ( in vitro ) of diaphragm samples for experiment 2. ................................ ................................ ................................ ...... 67 4 28 Skeletal muscle cross sectional area for experiment 2 .. ................................ ..... 68 4 29 Representative fluorescent staining of myosin heavy chain for experiment 2. ... 69 4 30 Mitochondrial respiratory function in per meabilized fiber from heart muscle for experiment 2.. ................................ ................................ ................................ 70 4 31 Mitochondrial respiratory function in permeabilized fiber from d iap hragm muscle for experiment 2 ................................ ................................ .................... 71 4 32 Rates of State 3 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experiment 2.. ................................ .................. 72 4 33 Rates of State 4 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experime nt 2. ................................ ................... 73

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10 4 34 Calpain activation in cardiac and skeletal muscle was determined via Western blotting for experiment 2.. ................................ ................................ ..... 74 4 35 Caspase 3 activation in the diaphragm was determined via Western blotting for experiment 2. ................................ ................................ ................................ 75 4 36 Apoptosis was determined by the terminal doxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for experiment 2. ................................ ........... 76 4 37 Representative images of sections statined using TUNEL assay for experiment 2.. ................................ ................................ ................................ ..... 77 4 38 Atrogin 1 was determined via Western blotting for experiment 2.. ...................... 78 4 39 MuRF 1 was determined via Western blotting for experiment 2.. ....................... 79 4 40 Cathepsin L was measured as a marker of increased degradation by the lysosomal proteolytic system for experiment 2.. ................................ ................. 80

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11 LIST OF ABBREVIATION S 4 HNE 4 Hydroxynoneal ATG Autophagy gene CSA Cross sectional area DAU Daunorubicin DOX Doxorubicin ET Ejection time FS Fractional shortening ICT Isovolumic contraction time IVRT Isovolumic relaxation time LV Left ventricle LVDd Left ventricle end diastolic diameter LVDs Left ventricle end systolic diameter MHC Myosin heavy chain MPI Myocardial performance index PWSV Posterior wall shortening velocity PWT Posterior ventri cular wall thickness RCR Respiratory control ratio ROS Reactive oxygen species SBDP Spectrin breakdown product SJA SJA 6017 SS 31 Szeto Schiller peptide 31 SWT Septal wall thickness

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DOXORUBICIN INDUCED CARDIAC AND SKELETAL MYOPATHY: THE ROLE OF MITOCHO NDRIAL REACTIVE OXYGEN SP EC I ES EMISSION AND CALPAIN ACTIVA TION By Kisuk Min December 2012 Chair: Scott K. Powers Major: Health and Human Performance Doxorubicin (DOX) is an antitumor agent used to treat a variety of cancers. Although DOX is a highly effective anti tumor treatment, DOX has been shown to induce detrimental effects in several tissues, including cardiac and skeletal muscle. At present, a complete understanding of the mechanisms responsible for DOX mediated cellular toxicity remains elusive. Although the exact causal mechanism of DOX mediated muscle myopath y is unclear, it appears that an increased cellular production of reactive oxygen spec ies (ROS) is a contributing factor. Specifically, it has been demonstrated that DOX induced ROS are generated by NAD(P) H oxidoreductases, such as mitochondrial NA DH dehydrogenase. Nonetheless, it remains unknown if mitochondrial ROS production is a requirement for DOX induced cardia c and skeletal muscle myopathy. Furthermore, treatment of animals with DOX activates the cysteine protease calpain in both cardiac and skeletal muscle fibers. This is significant because calpain activation can promote cellular injury. Nonetheless, it is unknown if calpain activation is a requirement for DOX induced cardiac and skeletal muscle myopathy. Therefore, this

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13 investigation tested the central hypothesis that mitoc hondrial ROS production plays a required role in DOX induced cardiac and skeletal muscle myopath y and that ROS mediated activation of calpain is a key contributor to DOX induced cardiac and skeletal muscle myopathy Our study reveals that mitochondria are the dominant source of oxidant production in the cardiac and skeletal muscle during DOX administration. Indeed, mitocho ndrial ROS emission is a required upstream signal to activate key proteases during DOX administration. F urthermore, our data also indicate that calpain activation is a requirement for DOX induced myopathy in the heart and skeletal muscles, and calpain inhi bition provides protection against DOX induced damage to cardiac and skeletal mu scles. Collectively, these findings demonstrate that mitochondrial ROS emission and calpain activation contribute to the development of DOX induced myopathy in cardiac and skel etal muscles.

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14 CHAPTER 1 INTRODUCTION Doxorubicin (DOX) is a highly effective antitumor agent wi dely used in cancer chemotherap y. Unfortunately, the clinic al application of DOX is limited by its adverse effects on several tissues and organs, including cardiac and skeletal muscle [ 1 3 ] Indeed, one of the most serious side effects of DOX is life threatening cardiac injury, including the development of c ardiomyopathy and ultimately congestive heart failure [ 4 5 ] In regard to the damaging effects on skeletal muscles, a recent report indicates that systemic DOX administration at clinical doses depresses skeletal muscle specific force production [ 6 ] Therefore, understanding the mechanism(s) responsible for DOX induced damage to ca rdiac and skeletal muscles is important. Althou gh experimentally unproven, DOX induced cellular damage and dysfunction could result from the oxidation of cellular components due to an increased production of reactive oxygen species (ROS). In this context, it has been established that DOX generates toxic free radicals due to its unique chemical structure [ 7 ] DOX induced free radicals are produced by NAD(P)H oxidoreductases, such as NADH dehydrogenase of the mitochondrial electron transport chain [ 8 9 ] and cytosolic xanthine oxidase [ 10 11 ] In reference to DOX induced oxidative stress, our prior work r evealed that mitochondria are the predominant source of oxidant production in cardiac and skeletal muscles during DOX admi nistration [ 12 13 ] In deed DOX t reatment significantly increases mitochondrial ROS production leading to mitochondrial dysfunction in cardiac m yocytes [ 12 ] In addition to DOX induced oxidative stress, DOX is also known to cause intracellular calcium (Ca 2+ ) overload [ 14 ] This could lead to activation of calcium

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15 dependent proteases (e.g., calpain) in ducing proteolysis of cellular proteins Specifically, calpain is a pr otease capable of degrading numerous muscle proteins that promote the release of myofilaments and we predict that calpain activation is a required step for DOX induced skeletal muscle atrophy [ 15 ] and cardiomyopathy [ 16 ] Therefore, these experiments tested the central hypothesis that mitoc hondrial ROS production plays a required role in DOX induced cardiac and skeletal muscle myopath y and that ROS mediated activation of calpain is a key contributor to DOX induced cardiac and skeletal muscle myopathy Our experiments provide a rigorous test of this prediction by addressing the following specific aims. Specific Aim 1: To determine if in creased mitochondrial ROS production is a requirement for DOX induced damage and contractile dysfunction in both the heart and skeletal muscle. It has been shown that mitochondria are a major source of oxidant production in both cardiac and skeletal muscle in animals treated with DOX. Using a mitochondrial targe ted antioxidant (SS 31), we prevent ed DOX induced mitochondrial ROS production in both cardiac and skeletal muscles to determine if increased mitochondrial ROS production is a requirement for DOX ind uced damage in cardiac and skeletal muscle. Specific Aim 2: To determine if calpain activation is a requirement for DOX induced damage to both cardiac and skeletal muscle Evidence suggests that DOX results in activation of calpain in both cardiac and ske letal muscle, leading to catabolic processes. Using a highly selective calp ain inhibitor (SJA6017), we determine d if DOX induced calpain activation is a requirement for DOX induced damage to both cardiac and skeletal muscle.

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16 CHAPTER 2 LITERATURE REVIEW Doxorubicin (DOX) is an anthracycline antibiotic and is the most effective anticancer drug used for treatment of many human neoplasms, including acute leukemias, lymphomas, stomach, breast and ovarian cancers [ 17 ] The first anthracyclines were isolated from the pigment producing Streptomyces peucetius in the 1960s and were named doxorubicin and daunorubicin (DAU) [ 18 ] While DAU has been shown to be highly effective against acute lymphoblastic and myeloblastic leukemias, DOX has been found to have a much broader anticancer spectrum, which includes numerous solid tumors in addition to hematological malignancies [ 7 ] Despite extensive clinical utilization, the mechanisms of action of anthracyclines in cancer cells remain a matter of controversy. Nonetheless, the following mechanisms have been proposed to explain the anticancer responses of DOX: 1) intercalation into DNA, leading to inhibited synthesis of macromolecules; 2) generation of free radicals, leading to DNA damage and/ or lipid peroxidation; 3) DNA binding and alkylation; 4) DNA cross linking; 5) interference with DNA unwinding or DNA strand separation and helicase activity; 6) direct membrane effects; 7) initiation of DNA damage via inhibition of topoisomerase II; and 8) induction of apoptosis in response to topoisomerase II inhibition [ 19 ] Unfortunately, in addition to its potent antitumor effect, it has been recognized that DOX causes adverse effects by inducing toxicity in both cardiac and skeletal muscle. This review will highlight our current knowled ge regarding the primary signaling pathways responsible for DOX induced toxicity in both cardiac and skeletal muscle. We begin with a discussion of the proposed mechanisms responsible for DOX mediated increases in oxidative stress in cardiac and skeletal m uscle fibers.

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17 Mechanisms for DOX induced Oxidative Stress Several studies suggest that increased oxidative stress plays an important role in the DOX induced development of cardiomyopathy and skeletal muscle wasting [ 20 23 ] The ability of DOX to induce reactive oxygen species ( ROS ) formation is predicted from its unique chemical structure, which contains a quinone moiety ( Figure 2 1) [ 7 ] One electron addition to the qui n one moiety in ring C of teracycle results in the formation of a semiquinone that quickly regenerates its parent qui n one by reducing oxygen to a superoxide anion. The cycle is supported by a number of NAD(P)H oxidoreductases such as mitochondrial NADH dehydrogenase which is an enzyme at complex I in the mitochondrial inner membrane [ 7 ] Hence, based on this mechanism of DOX induced free radical production, mitochondria could be an important source of ROS production during DOX administratio n. Indeed, our prior work shows DOX t reatment significantly increases mitochondri al ROS production in cardiac muscle, as well as oxidative damage in skeletal muscle [ 12 13 ] Mechanisms for DOX induced Cardia c and Skeletal Muscle Damage and Contractile Dysfunction DOX induced cytotoxicity leads to a progressive loss of muscle protein leading to c ardiac and skeletal muscle atrophy [ 13 ] This loss of muscle fiber protein occurs largely because of increases in proteolysis [ 13 ] DOX induced proteolysis in cardiac and skeletal muscle occurs via four major proteolytic systems: 1) Calpains; 2) Caspase 3; 3) Ubiquitin proteasome system; and 4) Autophagic/lysosomal sys tem. A brief overview of these proteolytic systems follows.

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18 Calpains Calpains (calpain I and II) are calcium dependent cysteine proteases that are present in all tissues [ 24 ] Specifically, calpains are capable of degrading numerous m uscle proteins (i.e. titin and nebulin) that promote the release of myofilaments and calpain activation appears to be a required step in remodeling of the myofibril [ 25 26 ] Furthermore, evidence reveals that calpain may work in conjunction with caspase 3 to degrade actomyosin complexes [ 15 ] Further new findings indicate th at calpain activation is a required initial step in both myocardial apoptosis and skeletal muscle degradation in a variety of catabolic conditions including DOX induced apoptotic pathways [ 12 14 ] Caspase 3 Caspase 3 is a cysteine protease that can be activated by a variety of signaling pathways including calpain mediated activation of pro apoptotic proteins [ 27 ] Importantly, caspase 3 has been shown to be activated during skeletal muscle wasting [ 28 29 ] and cardiomyopathy [ 30 ] and is capable of degradin g actomyosin complexes in skeletal muscles [ 28 ] In addition, caspase 3 activ ation is required for myonuclear apoptosis in both cardiac and skeletal muscle during DOX administration [ 12 13 ] In regard to a poptosis caspase 3 plays a major role in mitochondrial dependent apoptosis [ 28 ] Therefore, caspase 3 activation can contribute to DOX induced apoptosis in both cardiac and skeletal muscle. Ubiquitin Proteas ome System The proteasome is a large protein complex that is found in both the nucleus and the cytoplasm whose ma in f unction is to degrade damaged proteins [ 31 ] The total proteasome comp lex (26S proteasome) consists of a core proteasome subunit (20S)

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19 coupled with two regulatory complexes (19S) located at either end of the 20S core. Targeting proteins for degradation by the proteasome requires the coordinated reactions of three enzymes. In the first step, the ubiquitin activating enzyme (E1) activates ubiquitin by hydrolyzing ATP. Following activation, the ubiquitination of spe cific proteins is provided by one of a variety of ubiquitin conjugating enzymes (E2). In the last step, ubiquitin l igases (E3) recognize the specific protein to be ubiquitinated and catalyze the transfer of ubiquitin from E2 to the target protein. Among these enzymes, E3 ligases are critically important because they confer substrate specificity to this system [ 32 ] Although numerous E3 ligases exist, the muscle specif ic E3 ligases atrogin 1/MAFbx and MuRF1 have both been reported to be important in muscle protein degradation during many forms of muscle atrophy [ 33 34 ] The expression of atrogin 1/MAFbx and MuRF1 is regulated by two transcription factors, forkhead box O (Foxo) and NF kB [ 35 37 ] MuRF1 and atrogin 1/MAFbx activation result in the formation of a polyubiquitin chain on target proteins. The polyubiquitin tag allows for recognition and degradation of protein substrates by the proteasome [ 38 40 ] Autophagic/ Lysosomal Prote ases components through the lysosomal machinery. While the proteasome degrades myofibrillar and most soluble short lived proteins [ 41 42 ] autophagy lysosomal system degrades long lived proteins and organelles [ 43 44 ] In the autophagic process, targeted molecules and organelles in the cytoplasm are sequestered into double me mbrane vesicles called aut ophago somes, which are delivered to lysosomes forming autolysosomes. These cytosolic molecules are then degraded by lysosomal proteases (i.e. catheps ins). The induction of the autophagic/lysosomal system may be mediated

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20 by Foxo signaling which increases ge ne expression of required autophagy proteins [ 45 ] Although basal autophagy is important for maintaining cell survival by recycling old and damaged organelles and cytosolic proteins, excessive autophagy can induce pathological changes such as apop tosis, cell death, and atrophy [ 46 ] S ummary Doxorubicin is one of the most effective drugs widely used to treat a number of malignancies. However, DOX has been shown to induce deleterious effects in several tissues and organs, including cardiac and skeletal muscle. At present, a complete understandi ng of the mechanisms responsible for DOX medicated muscle toxicity remains elusive. In this regard, numerous studies have demonstrated that DOX induced cellular damage and dysfunction result from the oxidation of cellular components via elevated ROS produc tion. Our preliminary evidence suggests that mitochondria are a predominant source of DOX induced oxidative stress in both cardiac and skeletal muscle. Furthermore, administration of DOX has been reported to activate key proteases in cardiac and skeletal m uscle and our preliminary data suggests that calpain may play an important role in DOX induced cardiac and skeletal muscle damage and dysfunction.

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21 Figure 2 1. Proposed mechanisms of ROS formation by Doxorubicin [ 19 ]

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22 CHAPTER 3 MATERIALS AND METHODS This chapter will be divided into two sections. The first section will include the experimental design used for each of our experiments, which wer e designed to determine if increased mitochondrial ROS production and calpain activation contribute to DOX induced damage to both cardiac and skeletal muscle. In the second section, we will provide the methodological details associated with each experimental protoco l and measurement technique. Animals Animal Model Justification To address our first specific aim and establish whether increased mitochondrial ROS production is a requirement for DOX induced damage and cont ractile dysfunction in both heart and skeletal m uscle, we used adult female Sprague Dawley rats in experiment 1. Animals were 4 6 months old and ~300g at the time of sacrifice. The SD rat was chosen due to the similarities bet ween the rat and human muscle in both anatomical and physiological param eters [ 33 47 48 ] Animal Housing and Diet All animals were housed at the University of Florida Animal Care Services Center according to guidelines set forth by the Instit utional Animal Care and Use Committee. The Animal Care and Use Committee of the University of Florida approved these experiments. Animals were maintained on a 12:12 hour light dark cycle and provided food and water ad libitum throughout the experimental pe riod.

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23 Experiment 1 Experimental Design Aim 1 tested the hypothesis that mitochondrial ROS production contributes to DOX induced oxidative damage to both cardiac and skeletal muscle s (diaphragm, soleus, and plantaris muscles) In this experiment, adult fem ale SD rats were randomly assigned to one of t he following groups (n=8/group): 1) Control group injected with saline (CON); 2) DOX treatment group (DOX); 3) Control group treated with the mitochondrial targeted antioxidant SS 31 (SS 31); 4) DOX treatment with SS 31 (DOX+SS 31) (Figure 3 1). Experimental Protocol Anesthetized controls. Animals were initially anesthetized using 2 4% inhaled isoflurane. After re aching a surgical plane of anesthesia, the animals were sacrificed immediately and the heart, diaphragm, so leus, and plantaris muscles were be quickly removed and stored at 80 C for subsequent analyses. Doxorubicin administration. Animals assigned to DOX treatment received DOX hydrochloride (20 mg/kg of body weight). This dose of DOX is a human clinical dose of this drug that is pharmacologically scaled for use in rats [ 49 51 ] Saline was used as both the vehicle and the placebo. The heart, diaphragm, soleus, and plantaris muscles were excised for ana lysis 48 hours after injection. Mitochondrial targeted antioxidant (SS 3 1) administration. SS 31 is tetra peptide synthesized by Dr. Hazel H. Szeto in collaboration with Dr. Peter W. Schi l ler. The peptide SS 31 was chosen for this study because it targets mitochondrial inner membra ne and penetrates into cells [ 52 ] When the SS 31 peptide penetrates into mitochondria, tyrosine (Tyr) and dimethyltyrosine (Dmt) analogs from the peptides

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24 scavenge and diminish free radicals, such as hydrogen peroxide, hydroxy l radical, and peroxynitrite [ 52 54 ] Animals assigned to SS 31 treatment received daily intraperitoneal injections of SS 31 dissolved in PBS (3mg/kg) for 3 days. The first injection was one day before DOX administration. Saline was used as both the vehicle and the placebo. Statistical Analysis Group sample size was determined using a power analysis of preliminary data from our laboratory. Comparisons of functional measurements betwee n groups were made by two way ANOVA and comparisons of molecular and biochemistry measurements between groups were made by one way ANOVA, and when appropriate, a Tukey HSD test was performed. Significance was established at P<0.05. Experiment 2 Experimental Design Aim 2 tested the hypothesis that DOX is responsible for c alp a i n dependent proteolysis in both cardiac and skeletal muscle s (diaphragm, soleus, and plantaris muscles). In this experiment, adult female SD rats were randomly assigned to on e of the following groups (n=8/group) 1) Control group injected with saline (CON); 2) DOX treatment group (DOX); 3) Control group injected with calpain inhibitor SJA 6017 (SJA); and 4) DOX treatment with SJA 6017 (DOX SJA) (Figure 3 2). Experimental Protocol Anesthetized controls. Animals were initially anesthetized using 2 4% inhaled isoflurane. After reaching a surgical plane of anesthesia, the animals were sacrificed immediately and the heart, diaphragm, so leus, and plantaris muscles were be quickl y removed and stored at 80 C for subsequent analyses.

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25 Doxorubicin administration. Animals assigned to DOX treatment received DOX hydrochloride (2 0mg/kg of body weight). This do s e of DOX is a human clinical dose of this drug that is pharmacologically scal ed for use in rats [ 49 51 ] Saline was used as both the vehicle and the placebo. The heart, diaphragm, soleus, and plan taris muscles were excised for ana lysis 48 hours after injection with DOX Calpain inhibitor (SJA 6017 ) administration. SJA is a peptide inhibitor of calpain. While many of the catalytic site directed peptidyl calpain inhibitors lack intracellular specific ity, plasma membrane permeability, and potency, the permeability of SJA across cell membrane s is relatively high compared to other widely used inhibitor s [ 55 ] Numerous studies have investigated the therapeutic efficacy of SJA in vitro and in vivo and have shown the inhibitor to be effective in pr eventing apoptosis by inhibiting calpain activity [ 55 57 ] Animals assigned to SJA treatment received a daily intraperitoneal injection of SJA (3mg/kg) for 3 days. The first inject ion was performed one day before DOX administration. Saline was used as both the vehicle and the placebo. Statistical Analysis Group sample size was determined using a power analysis of preliminary data from our laboratory. Comparisons of functional measu rements between groups were made by two way ANOVA and comparisons of molecular and biochemistry measurements between groups were made by one way ANOVA, and when appropriate, a Tukey HSD test was performed. Significance was established at P<0.05.

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26 General M ethods Functional Measures Diaphragmatic Contractile Properties. T he entire diaphragm was removed and placed in a dissecting chamber containing a Krebs Hensleit solution equilibrated with 95% O 2 5% CO 2 gas. A muscle strip, including the tendinous attachments at the central tendon and rib cage was dissected from the midc ostal region. The strip was suspended vertically between two lightweight Plexiglas clamps with one end connected to an isometric force transducer (model FT 03, Grass Instruments, Quincy, MA) within a jacketed tissue bath. The force output was recorded via a computerized data acquisition system (Super Scope II, GW Instruments Somerville, MA; Apple Computer Cupertino, CA). The tissue bath was filled with Krebs Hensleit saline and the buffer was aerated with gas (95% O 2 5% CO2), pH was maintained at 7.4, and the osmolality of the bath was ~290 mosmol/kgH 2 O. After a 15 min equilibration period (25C), in vitro diaphragmatic contractile measur ements were made. The muscle strip was stimulated along its entire length with platinum wire electrodes (modified S48 stimulator, Grass Instruments) by using supramaximal (~150%) stimulation voltage to determine the optimum contractile length ( L o). L o was determined by systematically adjusting the length of the muscle using a micrometer while evoking single twitches. Thereafter, all contractile properties were measured isometrically at L o. To measure maximal isometric twitch force each strip was stimulated supramaximally with 120 V pulses at 1 Hz and to measure the force frequency response each strip was stimulated supramaximally with 120 V pulses at 15 160 Hz. The duration of each train was 500 ms to achieve a force plateau. Contractions were separated by a 2 min recovery period. For comparative purposes, diaphragmatic (bundles of fibers) force production was normalized as specific

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27 Po. The total muscle cross sectional area at right angles to the long axis was calculated by the following algorithm [ 58 ] Total muscle cross sectional area (mm 2 ) = [muscle mass/(fiber length x 1.056)], where 1.056 is the de nsity of muscle (in g/cm 3 ). Fiber length was expressed in centimeters measured at L o [ 59 ] Echocardiograms. Two dimensional and M mode echoca rdiographic measurements were performed on the posterior wall (PW) and left ventricle diameter of the heart during systole (LVDS) and diastole (LVDD) in the parasternal long axis view at the level of the pa pillary muscles. Left ven tricle systolic function was assessed by changes in fraction shorting (FS=LVDD LVDS/LVDD). Doppler measurement s of mitral inflow velocity were recorded in the apical four chamber view with the pulsed wave Doppler sample volume plac ed at the tips of the leaflets. Peak early ventricular filling (E) and atrial co ntraction (A) velocities were determined from these data. A change in the E/A ratio was used to assess left ventricle diastolic fu nction. All measurements were performed on thr ee distinct cardiac cycles and the values averaged at each time point. Histological Measures Myofiber Cross Sectional Area Tissues were removed and fixed in OCT and stored at 80C. On the day of analysis, sections from frozen diaphragm, soleus, and plan taris samples were cut at 10 microns with a cryotome (Shandon Inc., Pittsburg, PA) and allowed to air dry at room temperature for 30 minutes. Sections were stained for dystrophin, myosin heavy chain (MHC) I and MCH type IIa proteins for fiber cross section al area (CSA) as previously described [ 29 ] CSA was determined using Scion software (NIH). Apoptosis. Myo nuclear apoptosis was determined by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) using a histochemical fluorescent detection kit

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28 Shandon Cryotome cryostat (Life Sciences Internati onal; England) from embedded muscle tissue s Sections were fixed using in a 4% formaldehyde solution, washed, and permeabilized with 0.1% Triton X 100 in 0.1% sodium citrate solution. For identification of cell membranes, tissues were incubated with a rabbit anti dystrophin antibody (Thermo Scientific; Freemont, CA), and secondary conjugated to Rhodamine Red (Invitrogen; Eugene, OR). Tissue sections were then incubated with the TUNEL enzyme label solution and sealed with a Vectashield DAPI mounting medium for detection of nuclei (Vector Laboratories; Burlingame, CA). TUNEL stained tissue sections were imaged using a Zeiss fluorescent microscope (Thornwood, NY). Fluorescent images for DAPI (nuclei), Rhodamine (dystrophin), an d FITC filters (TUNEL positive) were combined using IPLab software (Scanalytics, Inc.; Fairfax, VA). TUNEL positive nuclei were counted and normalized to tissue cross sectional area. Biochemical Measures Western Blot Analysis. Protein abundance was determi ned in whole heart, diaphragm, sol eus, and plantaris samples via W estern blot analysis. Briefly, tissue samples were homogenized 1:10 (wt/vol) in 5mM Tris (pH 7.5) and 5 mM EDTA (pH 8.0) with a protease inhibitor cocktail (Sigma, St. Louis, MO) and centrif uged at 1,500g for 10 min at 4C. After the resulting supernatant was collected, the protein content was assessed by the method of Bradford (Sigma). Proteins from the supernatant fraction were separated via polyacrylamide gel electrophoresis via 4 20% grad ient polyacrylamide gels containing 0.1% SDS for ~1 h at 200 V. After electrophoresis, the proteins were transferred to nitrocellulose membranes and incubated with primary antibodies directed against proteins of interest. Following incubation with primary

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29 antibodies, membranes were washed extensively with PBS Tween and then incubated with secondary antibodies (GE Healthcare, Piscataway, NJ). After being washed, a chemiluminescent system was used to detect labeled proteins (GE Healthcare). Membranes were dev eloped using autoradiography film and images of the film were captured and analyzed using the 440CF Kodak Imaging System (Kodak, New Haven, CT). Alpha tubulin was analyzed to provide a control for protein loading. Mitochondrial Measures Preparation of Per meabilized Muscle Fibers Approximately 25 mg of costal heart, diaphragm, soleus, and plantaris muscles were dissected and placed on a plastic Petri dish containing ice cold buffer X ( 60 mM K MES, 35 mM KCl, 7.23 mM K 2 EGTA, 2.77 mM CaK 2 EGTA, 20 mM imidazol e, 0.5 mM DTT, 20 mM taurine, 5.7 mM ATP, 15 mM PCr, and 6.56 mM MgCl 2 pH 7.1 ). The muscle was trimmed of connective tissue and cut down to fiber bundles (4 8 mg wet wt). The muscle fiber bundles were gently separated in ice cold buffer X to maximize surf ace area of the fiber bundle. To permeabilize the myofibers, each fiber bundle was incubated in ice cold buffer X saponin for diaphragm muscle on a rotator for 30 min at 4C. T he permeabilized muscle bundles were then washed in ice cold buffer Z ( 110 mM K MES, 35 mM KCl, 1 mM EGTA, 5 mM K 2 HPO4, and 3 mM MgCl 2 0.005 mM glutamate, and 0.02 mM malate and 0.5 mg/ml BSA, pH 7.1 ). Mitochondrial Respiration. Respiration was measured polarographically in a respiration chamber maintained at 37C (Hanstech Instrumnets, United Kingdom). After the respiration chamber was calibrated, permeabilized fiber bundles were incubated with 1 ml of respiration buffer Z containing 20mM creatine to sa turate creatine kinase.

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30 Flux through complex I was measured using 5mM pyruvate and 5mM malate. The ADP stimulated respiration (state 3) was initiated by adding 0.25 mM ADP to the respiration chamber. Basal respiration (state 4) was determined in the presen oligomycin to inhibit ATP synthesis. The respiratory control rati o (RCR) was calculated by dividing the state 3 by state 4 respiration. Mitochondrial ROS Emission. Mitochondrial ROS emission was determined using Amplex Red (Molecular Probes, Eugene, OR). Details of this assay have been described previously [ 60 ] Mitochondrial ROS production was measured using the creatine kinase energy clamp technique to maintain respiration at steady state using previo usly described methods [ 61 ]

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31 Functional Measurements: Molecular and Biochemistry: Histological Measure: Mitochondrial respiration Oxidative production: ROS production and 4 HNE sectional area Cardiac fractional shortening Protease activity: Calpain and caspase 3 Myocardial performance index MuRF 1 and atrogin 1 Skeletal muscle force frequency curve Figure 3 1. Experimental animal design used to determine if increased mitochondrial ROS production is a requirement for DOX induced damage and contractile dysfu n ction in both cardiac and skeletal muscle. Sprague Dawley Rat (n=8/group) CON DOX SS 31 DOX+SS31

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32 Functional Measurements: Molecular and Biochemistry: Histological Measure: Mitochondrial respiration Oxidative production: ROS production and 4 HNE sectional area Cardiac fractional shortening Protease activity: Calpain and caspase 3 Myocardial performance index MuRF 1 and atrogin 1 Skeletal muscle force frequency curve Lysosomal protease: Cathepsin Figure 3 2 Experimental animal design used to determine if calpain activation is a requirement for DOX induced damage to both cardiac and skeletal muscle. Sprague Dawley Rat (n=8/group) CON DOX SJA DOX+SJA

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33 CHAPTER 4 RESULTS Experiment 1 : Systemic Response to Doxorubicin Administration Experiment 1 was designed to determine if increased mitochondrial ROS production is a requirement for DOX induced myopathy in bot h cardiac and skeletal muscles. Animals used in this experiment were 4 6 months of age and prior to DOX administration no sig nificant differences existed in body weight between groups. However, body weight of the DOX group was significantly decreased at 24 hours and 48 hours after DOX administration (Table 4 1 Figure 4 1 ). Cardiac Function Changes in cardiac weight in response to doxorubicin administration We measured the heart weight, left ventricular weight, left ventricle to heart weight ratio and heart to body weight ratio to determine if DOX administration results in cardiac weight changes In this regard, all animals in the DOX group demonstrated a significant reduction in all weight parameters measured compared to CON animals. Importantly, treatment of DOX animals with SS 31 attenuated the changes to left ventricular weig ht and to the lef t ventricle to heart weight ratio (P<0.05) (Table 4 2). Cardiac g eometry and f unction To determine the effect of SS 31 on cardiac function during DOX administration, echocardiographic measures were assessed. Cardiac muscle dimensions obtained via this meth od are shown in Table 4 3. DOX administration resulted in a thinning of the septal walls (SWT) and posterior ventricular walls (PWT) during both systole and diastole (P<0.05). In addition, p osterior wall shortening velocity (PWSV) was significantly lower i n the DOX group Importantly, treatment of control animals with SS

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34 31 resulted in no changes in cardiac size or function compared to CON animals. Finally treatment with SS 31 in combination with DOX prevented against DOX induced thinning of the septal wal l left ventricular wall and decrease s in PWSV To assess the effect of SS 31 on DOX induced left ventricle systolic dysfunction, fractional shorten ing (FS) was measured by M mode echocardiography obtained for measures of LV end systolic diameter (LVDs), and LV end diastolic diameter (LVDd) (Figure 4 2). FS was calculated as (LVDd LVDs)/LVDd. DOX administration resulted in a significant reduction of FS com pared to both CON and DOX+SS 31 (P<0.05) (Figure 4 3). Additionally, there was no difference in FS bet ween CON and control animals w ith SS 31. To examine if treatment with SS 31 protects against DOX induced impairment of both systo lic and diastolic function the myocardial performance index (MPI) was assessed by Doppler images (Figure 4 4). MPI was calculated as (ICT+IVRT)/ET, where ICT is isovolumic contraction time, IVRT is isovolumic relaxation time and ET is ejection time. DOX administr ation significantly increased MPI compared to both CON and DOX+SS 31 (P<0.05) (Figure 4 5). Note that no differ ence s existed in MPI between CON and SS 31 animals. Diaphragm Contractile Function Force frequency response The diaphragm force frequency response was measured in our experimental groups to determine the effectiveness of SS 31 treatment in maintaining di aphragm contractile function during DOX administration. The mean specific force frequency responses from all groups are shown in Figure 4 6. DOX administration resulted in a significant reduction (P<0.05) in the specific force of the diaphragm in the DOX g roup, compared to the CON and SS 31 group at all stimulation

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35 frequencies. Note, h owever that treatment with SS 31 protected against DOX induced diaphragm contractile dysfunction at stimulation frequencies greater than 30Hz. Skeletal Muscle Structure Cros s sectional area Myofiber c ross sectional area (CSA) was evaluated in diaphragm, soleus and plantaris skeletal muscles to determine the role of mitochondrial ROS production on DOX induced skeletal muscle atrophy. Compared to CON, we observed a significan t (P<0.05) decrease in muscle Type I, Type IIa and Type IIx/b fiber CSA in the DOX group following DOX administration (Figure 4 7, Figure 4 8) In addition, no difference s existed in fiber CSA between CON and control animals with SS 31. Finally, treatment with SS 31 resulted in protection against DOX induced atrophy of Type I, Type IIa and Type IIb/x fiber types in all muscles Mitochondrial Function Respiratory control ratio To determine whether treatment with SS 31 protects both cardiac and skeletal muscle mitochondria from DOX induced mitochondrial uncoupling, we measured the mitochondrial respiratory control rati o (RCR). As shown in Figure 4 9 and Figure 4 10 DOX administration significantly reduced the RCR in cardiac and skeletal muscle, compared to CON and DOX+SS 31 (P<0.05). Additionally, t here was no difference in RCR between control animals with SS 31 and the CON group. Importantly, treatment with SS 31 prevented DOX induced mitochondrial uncoupling. Redox Balance Mit ochondrial reactive oxygen species emission Mitochondria have been shown to be the primary source of DOX induced reactive oxygen species (ROS) production in cardiac and skeletal muscles [ 12 62 ] Therefore,

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36 w e determined the effect of SS 31 administration on DOX induced ROS emission from cardiac and skeletal muscles by measuring mitochondrial ROS emission. In this regard our data shows mitochondrial H 2 O 2 release is increased in car diac and skeletal muscle with DOX administration and treatment of animals with SS 31 effectively inhibits mitochondrial ROS emission in cardiac and skeletal muscle during both state 3 and state 4 respiration (P <0.05) (Figure 4 11, Figure 4 12 ). 4 HNE 4 hydroxynoneal (4 HNE) was measured as an indicator of lipid peroxidation in both the heart and skeletal muscles to determine if mitochondrial ROS production is responsible for increased oxidative stress during DOX adm inistration. Compared to CON DOX administration resulted in a significant increase in 4 HNE modified proteins in cardiac and skeletal muscles. However, treatment of animals with SS 31 protected the heart and skeletal muscles against the DOX induced in crease s in 4 HNE. These findings indicate that increased mitochondrial ROS production is a requirement for DOX induced oxidative damage in cardiac and skele tal muscle (P<0.05) (Figure 4 13 ). Proteolytic Activity Calpain To determine whether DOX induced inc reases in mitochondrial ROS production are required to activate calpain in cardiac and skeletal muscle, cal pain activity was evaluated by W estern blot for both the activ e band of calpain 1 (Figure 4 14 ) as well as the calpain specific spectrin breakdown pr oduct (SBDP) (Figur e 4 15 ). Our resu lts show that calpain activity wa s increased in cardiac and skeletal m uscle during DOX administration whereas treatment with SS 31 prevented the increase in calpain activity in both the cardiac and skeletal muscle (P<0. 05).

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37 Caspase 3 Caspase 3 is a protease that is capable of degrading intact actomyosin proteins and it also plays a key role in promoting myonuclear apoptosis in muscle fibers. Compared to control animals, DOX administration resulted in a significant increa se in both cleaved (active) caspase 3 (Figure 4 16 ) and in the protein levels of the caspase 3 specific SBDP in cardiac and skeletal muscle (P<0.05) (Figure 4 17 ). Treatment with SS 31 prevented DOX induced increases in caspase 3 in both the cardiac muscle as well as the diaphragm, soleus and plantaris muscles, compared to CON In addition, TUNEL staining was used as a biomarker of myonuclear apoptosis. DOX administration resulted in a significantly higher number of TUNEL positive nuclei in DOX groups in both cardiac and skeletal muscles. However, treatment with SS 31 prevented DOX induced increases in myonuclear apoptosis in the cardiac and skelet al muscles (P<0.05) (Figure 4 18, Figure 4 19 ) Ubiquitin proteasome system To determine whether DOX induced increases in mitochondrial ROS production are responsible for activation of the muscle specific E3 ligases protein content of atrogin 1 and MuRF 1 was measured Our results demonstrate that DOX administration results in increase d protein levels of both Atrogin 1 (Figure 4 20 ) and MuRF1 (Figure 4 21 ) in DOX animals in both heart and skeletal muscle However, treatment with SS 31 prevented the DOX induced increase in protein abundance of atrogin 1 and MuRF1 in all muscle groups Experiment 2 : Systemic Response to Doxorubicin Administration Experiment 2 investigated the role that c alpain plays in DOX induced cardia c and skeletal muscle myopathy. Animals used in this experiment were 4 6 months of age and

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38 no significant differences existed in initial animal body weight between groups. However, DOX significantly decreased animal body w eight at 24 hours and 48 hours after DOX administration in both the DOX and DOX+SJA groups (Table 4 4 Figure 4 22 ). Cardiac Function Changes in cardiac weight in response to doxorubicin administration We measured the heart weight, left ventricular weight, left ventricle to heart weight ratio, and heart to body weight ratio DOX administration significantly decreased heart weight, left ventricular weight and heart to body weight ratio, compared to CON and SJA groups. Importantly, treatment of DOX animals wi th SJA prevented DOX induced decreases in the heart weight and left ventricular weight after 48 hours of DOX administration (P<0.05) (Table 4 5). Cardiac geometry and function To determine the eff ect of calpain inhibition on cardiac morphologic changes during DOX administration, we measured c ardiac dimensions using echocardiography ( Table 4 6 ) DOX administration resulted in a thinning of the septal walls (SWT) and posterior ventricular walls (PWT) during both systole and dia stole in DOX groups (P<0.05), compared to CON and SJA groups. PWSV was also significantly lower in the DO X group. Treatment with SJA prevent ed DOX induced thinning of the septa l walls and posterior ventricular wal ls during systole and decreases in PWSV I n addition, there was no difference in cardiac wall thickness in control animals with SJA, compared to the CON group. To assess the effect of calpain inhibition on DOX induced left ventricle systolic dysfunction, fractional shortening (FS) was measured by M mode echocardiography

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39 (Figure 4 23 ). DOX administration promoted a significant reduction of FS in the DOX group, compa red to CON and DOX+SJA (P<0.05) (Figure 4 24 ). Additionally, no difference s existed in FS between the control animals with SJA and the CON group. However, FS of DOX animals treated with SJA remained significantly low er than the SJA group. To determine whether prevention of calpain activation via treatment with SJA can protect against DOX induced impairment of cardiac function, we determin ed the myocardial performance index (MPI) using Doppler images (Figure 4 25 ). DOX administration significantly impaired myocardial performance (i.e., increased MPI ) compared to CON (P<0.05) (Figure 4 26 ). In addition, no difference s existed in MPI between CON and SJA animals. However, prevention of calpain activation did not provide significant protection ag ainst DOX induced increased MPI Diaphragm Contractile Function Force frequency response The diaphragm force freque ncy response was measured in these experiments to determ ine if calpain inhibition protects against DOX induced diaphragm contractile dysfunction. Compared to CON, DOX administration resulted in a significant reduction (P<0.05) in the specific force production of the diaphragm in t he DOX group at all stimulation frequencies ( Figure 4 27 ) However, prevention of DOX induced calpain activation in the DOX+SJA group protected the diaphragm against DOX mediated decreases in muscle force production. Finally, treatment of control animals w ith SJA did not alter diaphragm force production at any stimulation frequency.

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40 Skeletal Muscle Structure Cross sectional area Myofib er c ross sectional area (CSA) was evaluated to determine the role of active calpain on DOX induced skeletal muscle atroph y. In diaphragm, soleus and plantaris muscles we observed a significant (P<0.05 ) decrease in muscle Type I, Type IIa and Type IIx/ b fiber CSA in DOX group compared to CON (Figure 4 28, Figure 4 29 ). In addition, no difference s existed in fiber C SA in control animals treated with SJA compared to CON grou p. Finally, treatment with SJA resulted in protection against DOX induced atrophy of Type I, Type IIa and Type IIb/x fiber types. Mitochondrial Function Respiratory control ratio To determine w hether the effect of inhibition of calpain protects mitochondria from DOX induced mitochondrial uncoupling, we measured the mitochondrial respiratory control ratio (RCR). As show n in Figure 4 30 and Figure 4 31 DOX administration significantly reduced the RCR in cardiac and skeletal muscle compared to CON (P<0.05). Importantly, treatment with SJA prevented DOX induced mitochondrial uncoupling. Additionally, there was no difference in RCR in control animals treated with SJA, compared to the CON group. Redox Balance Mitochondrial ROS e mission Our data indicate that compared to CON, DOX administration results in a significant increase in H 2 O 2 emission in mitochondria within permeabilized cardiac and skeletal muscle fiber bundles Interestingly, compared to the DOX group, treatment with SJA resulted i n a complete attenuation of the DOX induced increase d mitochondrial ROS emission in cardiac and skeletal muscle s (P<0.05) (Figure 4 32 and 4 33 ).

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41 Proteolytic Activity Treatment wit h SJA prevents DOX induced Calpain activation To investigate the role of calpain signaling in cardiac and skeletal muscles during DOX administration, we used a highly selective pharmacological inhibitor of calpain activity (SJA6017). Calpain activity in cardiac and skeletal muscles was measured by protein abundance of active calpain detected via W estern blotting. Our data indicated that DOX administration resulted in a significant increase in the pre sence of active calpain 1 in both heart and skeletal muscles (P<0.05) (Figure 4 34 ). However, treatment with SJA prevented DOX induced increases in calpain activity in cardiac and skeletal muscles. Therefore, our results indicated that SJA selectively inhibited calpain activity i n the heart and skeletal muscle. Caspase 3 Caspase 3 has bee n shown to degrade myofibrillar proteins and play an important role in DOX induced muscle myopathy [ 12 13 ] Compared to control animals, DOX administra tion resulted i n a significant increase in cleaved (active) caspase 3 in the cardiac and skelet al muscles (P<0.05) (Figure 4 35 ). Importantly, compared to CON, treatment with SJA prevented DOX induced increases in c aspase 3 in both c ardiac and skeleta l muscles DOX administration resulted in a significantly higher number of TUNEL positive nuclei in both cardiac and skeletal muscles. However, treatment with SJA prevented DOX induced increases in myonuclear apoptosis in the cardiac and skeletal muscles (P<0.05) (Figure 4 36, Figure 4 37). Ubiquitin proteasome system To determine whether DOX induced increases in calpain activation are responsible for activation of the muscle specific E3 ligases, protein levels of atrogin 1 and MuRF 1

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4 2 were measured in both heart and skeletal muscles Our results demonstrate that DOX administration results in an increase in protein levels of Atrogin 1 (Figure 4 38) and MuRF1 (Figure 4 39) in DOX treated animals in both heart and skeletal muscle. However, tre atment with SJA prevented the DOX induced increase in protein abundance of atrogin 1 a nd MuRF1 in all muscles Lysosomal Protease Cathep sin L The l ysosomal proteolytic system is comprised of a system of proteins that work together to degrade damaged cyto solic proteins and organelles. In this regard, we measured protein abundance of cathepsin L to determine the contribution of the lysosomal system to DOX induced myop a thy. Our data reveal that the protein expressi on of cathepsin L wa s significantly increase d in both cardiac and skeletal muscles following DOX administration. Importantly, inhibition of ca lpain signaling prevented the DOX induced increase in cathepsin L protein levels in the heart and skelet al muscles (P<0.05) (Figure 4 40 ).

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43 Table 4 1. Body weight change s for experiment 1. Hours CON (g) DOX (g) SS 31 (g) DOX+SS31 (g) 0 309.78.08 313.89.67 307.93.04 309.56.48 24 310.38.63 297.410.08 302.42.91 300.67.66 48 312.07.55 288.19.93 307.32.25 295.5+7.28 Values are expressed as mean SE. Sign ificantly different versus CON (P<0.05). Table 4 2. Echocardiographic variables for experiment 1. CON DOX SS 31 DOX+SS31 Heart Weight ( m g) 813.625.2 690.916.7* 806.428.4 7 30.419.9# LV weight ( m g) 184.57.00 143.92.99** 172.67.48 161.45.48 LV/Heart 0.230.004 0.210.003* 0.210.006 0.220.002 Heart/BW (mg/g) 2.630.05 2.210.02* 2.620.08 2.360.05# Values are expressed as mean SE. Signific antly different versus CON. Significantly different versus CON and DOX+SS 31. # Significantly different versus SS 31 (P<0.05). Table 4 3. Echocardiographic parameters for experiment 1. CON DOX SS 31 DOX+SS31 SWTd (mm) 1.460.02 1.280.06** 1.460.02 1.450.03 SWTs (mm) 2.720.05 2.080.06** 2.550.03 § 2.530.04 PWTd (mm) 1.430.04 1.240.04** 1.480.03 1.450.03 PWTs (mm) 2.540.05 2.010.07** 2.580.07 2.400.04 # PWSV (cm/sec) 31.340.37 22.060.84** 30.330.82 29.090.60 Values are expressed as mean SE. ** Significantly different versus CON and DOX+SS 31. § Significantly different versus CON # Sign ificantly different versus SS 31 (P<0.05). Table 4 4. Body weight changes for experiment 2 Hours CON (g) DOX (g) SJA (g) DOX+SJA (g) 0 275.53.36 293.310.78 277.54.91 279.36.02 24 273.14.76 280.811.32 276.85.33 264.84.76 48 274.04.20 276.611.05 276.15.32 263.04.97 Values are expressed as mean SE.

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44 Table 4 5. Echocardiographic variables for experiment 2 Hours CON DOX SJA DOX+SJA Heart Weight ( m g) 732.122.6 659.415.0* 738.020.65 719.923.1 LV weight ( m g) 161.36.56 144.14.23* 162.85.49 158.04.36 LV/Heart 0.220.004 0.220.008 0.220.003 0.220.004 Heart/BW (mg/g) 2.660.08 2.260.04* 2.660.04 2.390.07 # Values are expressed as mean SE. Significantly different versus CON. # Sig nificantly different versus SJA (P<0.05). Table 4 6. Echocardiographic parameters for experiment 2 CON DOX SJA DOX+SJA SWTd (mm) 1.540.04 1.340.06* 1.560.03 1.430.03 # SWTs (mm) 2.760.08 2.290.04** 2.630.07 2.540.04 PWTd (mm) 1.630.05 1.320.06* 1.560.03 1.430.03 # PWTs (mm) 2.660.04 2.190.04** 2.680.05 2.360.05 # PWSV (cm/sec) 30.231.28 22.69073* 29.082.51 25.530.57 Values are expressed as mean SE. Significantly different versus CON. ** Significantly different versus CON and DOX+SJA # Sig nificantly different versus SJA (P<0.05).

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45 A B C Figure 4 1 Body weight changes for experiment 1. Values are mean SE. A) Body w eight before DOX administration. B) Body weight changes after 24 hours of DOX administration C) Body weight changes after 48 ho urs of DOX administration ** Significantly different versus CON and DOX+SS 31 # Significantly different versus DOX+SS 31 (P<0.05)

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46 A B C D Figure 4 2 Representative M mode echocardi ograms for experiment 1. A) CON. B) DOX. C) SS 31. D) DOX+SS 31. Figure 4 3 Fractional shortening for experiment 1. Values are mean SE. ** Significantly different versus CON and DOX+SS 31. # Significantly different v ersus SS 31 (P<0.05).

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47 A B C D Figure 4 4 Representative Doppler echocardiograms for experiment 1. A) CON B) DOX. C) SS 31. D) DOX+SS 31. Figure 4 5 Myocardial Performance Index for experiment 1. Values are mean SE. ** Significantly diff erent versus CON and DOX+SS 31. # Significantly different versus SS 31 (P<0.05).

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48 Figure 4 6 Diaphragm force frequency response ( in vitro ) of diaphragm samples for experiment 1. Values are mean SE. DOX Significantly different versus CON. ** DOX Significantly different versus CON and DOX+SS 31. # DOX+SS 31 significantly different versus SS 31 (P<0.05).

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49 A B C Figure 4 7 Skeletal muscle cross sectional area for experiment 1. Values are mean SE. A) Diaphragm. B) Soleus. C) Plantaris ** S ignificantly different versus CON and DOX+SS 31 (P<0.05).

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50 A B C Figure 4 8 Representative fluorescent staining of myosin heavy chain for experiment 1. A) Diaphragm. B) Soleus. C) Plantaris

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51 A B C Figure 4 9. M itochondrial respiratory functio n in permeabilized fiber from Heart muscle for exper iment 1. A) State 3 Respiration. B) State 4 Respiration. C) Respiratory Control Ratio. Values are mean SE. Significantly different versus DOX+SS 31. Significantly diff erent versus CON and DOX+SS 31 (P<0.05).

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52 A B C Fig ure 4 10 M itochondrial respiratory function i n permeabilized fiber from diaphragm muscle for experiment 1 A) State 3 Respiratio. B) State 4 Respiration. C) Respiratory Control Ratio. Values are mean SE. ** Significantly different versus CON and DOX+SJA. # Significantly different versus S S 31 (P<0.05).

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53 A B C D Figure 4 11 Rates of State 3 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experiment 1. Values are mean SE. A) Heart. B) Diaphrag m. C) Soleus. D) Plantaris. ** Significantly different versus CON and DOX+SS 31 (P<0.05).

PAGE 54

54 A B C D Figure 4 12 Rates of State 4 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experiment 1. Values are mean SE. A) Heart. B) Dia phragm C) Soleus. D) Plantaris ** Significantly different versus CON and DOX+SS 31 (P<0.05).

PAGE 55

55 A B C D Figure 4 13 The levels of 4 hydroxynonenal (4 HNE) were analyzed as an indicator o f lipid pero xidation via W estern blotting for experiment 1. Values are mean percentage change SE A) Heart. B) Diaphr agm. C) Soleus. D) Plantaris. Significantly different versus CON and DOX+SS31 (P<0.05).

PAGE 56

56 A B C D Figure 4 14 Calpain activation in cardiac and skeletal muscle was determined via Western blotting for experiment 1. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Significantly different versus CON. ** Significantly different CON and DOX+SS 31 (P<0.05).

PAGE 57

57 A B C D Figure 4 15 Calpain specific spectrin breakdown product (SBDP) was determined for experiment 1. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Significantly different versus CON. ** Significantly different CON and DOX+SS 31 (P<0 .05).

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58 A B C D Figure 4 16 Caspase 3 activation in the diaphragm was determined via Western blotting for experiment 1. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Values are mean SE. Significantly different versus CON. ** Significantly diffe rent versus CON and DOX+SS 31 (P<0.05).

PAGE 59

59 A B C D Figure 4 17 Caspase 3 specific spectrin breakdown product was determined (SBDP) for experiment 1. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Significantly different versu s CON. ** Significantly different versus CON and DOX+SS 31 (P<0.05).

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60 A B C D Figure 4 18 Apoptosis was determined by the terminal doxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for experiment 1. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. ** Significantly different versus CON and DOX+SS 31 (P<0.05).

PAGE 61

61 A B C D Figure 4 19 Representative images of sections statined using TUNEL assay for experiment 1 A) Heart. B) Diaphragm. C) Soleus. D) Plantaris

PAGE 62

62 A B C D Figure 4 20 Atrogin 1 was determined via Western blotting for experiment 1. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. ** Significantly different versus CON and DOX+SS 31 (P<0.05).

PAGE 63

63 A B C D Figure 4 21 MuRF 1 was determined via Western blotting for experiment 1. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris Significantly different versus CON, ** Significantly dif ferent versus CON and DOX+SS 31, # Significantly different versus CON and DOX (P<0.05).

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64 A B C Figure 4 22 Body weight changes for experiment 2 Values are mean SE. A) Body w eight before DOX administration. B) Body weight changes after 24 hours of DOX administration. C) Body weight changes after 48 hours of DOX administratio n Significantly different versus CON # Significantly different versus SJA. (P<0.05).

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65 A B C D Figure 4 23 Representative M mode echocardi ograms for experiment 2. A) CON. B) DOX. C) S JA. D) DXO+SJA. Figure 4 24 Fractional shortening for experiment 2. Values are mean SE. ** Significantly different versus CON and DOX+SJA # Significantly different versus CON (P<0.05).

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66 A B C D Figure 4 25 Representative Doppler echocardi ograms for experiment 2. A) CON. B) DOX. C) SJA. D) DOX+SJA. Fi gure 4 26 Myocardial Performance Index for experiment 2. Values are mean SE. *Significantly different versus CON. # Significantly different versus SJA (P<0.05).

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67 Figure 4 27 Diaphragm force frequency response ( in vitro ) of diaphragm samples for experiment 2. Values are mean SE. DOX Significantly different versus CON. ** DOX Significantly different versus CON and DOX+SJA. # DOX+SJA significantly different versus SJA (P< 0.05).

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68 A B C Figure 4 28 Skeletal muscle cross sectional area for experiment 2 Values are mean SE. A) Diaphragm. B) Soleus. C) Plantaris. S ignificantly different versus CON ** S ignificantly different versus CON and DOX+SJA (P<0.05).

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69 A B C Figure 4 29 Representative fluorescent staining of myosin heavy chain for experiment 2 A) Diaphragm. B) Soleus. C) Plantaris

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70 A B C Fig ure 4 30 M itochondrial respiratory function in permeabili zed fiber from h eart muscle for exper iment 2. A) State 3 Respiration. B) State 4 Respiration. C) Respiratory Control Ratio. Values are mean SE. Significantly different versus CON. ** Significantly different versus CON and DOX+SJA (P<0.05).

PAGE 71

71 A B C Figure 4 31 M itochondrial respiratory function in permeabilized fiber fr om diaphragm muscle for experiment 2 A) State 3 Respiration. B) State 4 Respiration. C) Respiratory Control Ratio. Values are mean SE. Significantly different versus CON. ** Significantly different CON and DOX+SJA (P<0.05).

PAGE 72

72 A B C D Figure 4 32 Rates of State 3 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experiment 2. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. ** Significantly different versus CON and DOX+SJA ( P<0.05).

PAGE 73

73 A B C D Figure 4 33 Rates of State 4 hydrogen peroxide release (H 2 O 2 ) release from permeabilized muscle fibers for experiment 2. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. ** Significantly different versus CON and DOX+SJA # Significantly different versus CON (P<0.05).

PAGE 74

74 A B C D Figure 4 34 Calpain activation in cardiac and skeletal muscle was determined via Western blotting for experiment 2. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plan taris. Significantly different versus CON. ** Significantly different CON and DOX+SJA (P<0.05).

PAGE 75

75 A B C D Figure 4 3 5 Caspase 3 activation in the diaphragm was determined via Western blotting for experiment 2. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Values are mean SE. Significantly different versus CON. ** Significantly different versus CON and DOX+SJA. # Significantly different versus CON (P<0.05).

PAGE 76

76 A B C D Figure 4 36 Apoptosis was determin ed by the terminal doxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for experiment 2 Values are mean SE. A) Heart. B) Dia phragm. C) Soleus. D) Plantaris. Significantly different versus CON, ** Significantly different versus CON and DOX +SJA, # Significantly different versus CON and DOX+SJA (P<0.05).

PAGE 77

77 A B C D Figure 4 37 Representative images of sections statined using TUNEL assay for experiment 2 A) Heart. B) Diaphragm. C) Soleus. D) Plantaris.

PAGE 78

78 A B C D Figure 4 38 Atrogin 1 was determined via Western blotting for experiment 2. Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Significantly different versus CON, ** Significantly di fferent versus CON and DOX+SJA # Significantly different versus CON and DOX+SJA (P<0.05).

PAGE 79

79 A B C D Figure 4 39. MuRF 1 was determined via Western blotting for experiment 2 Values are mean SE. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. ** Significantly di fferent versus CON and DOX+SJA (P<0.05).

PAGE 80

80 A B C D Figure 4 40 Cathepsin L was measured as a marker of increased degradation by the lysosomal proteolytic sy stem for experiment 2. A) Heart. B) Diaphragm. C) Soleus. D) Plantaris. Values are mean SE. ** Significantly different versus CON and DOX+SJA. # Significantly different versus CON (P<0.05).

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81 CHAPTER 5 DISCUSSION Overview of Principal Findings These experiments prov ide new and important information regarding the mechanisms responsible for DOX induced myopathy in both card iac and skeletal muscle In this regard we tested the hypothesis that mitochondrial ROS emission is a requirement for DOX induced damage to cardiac and skeletal muscle and that prevention of mitochondrial ROS production is sufficient to attenuate DOX induced muscular damage O u r results support this hypothesis and demonstrate that mitochondria are the dominant source of DOX induced oxidant production and that mitochondrial ROS emission is a required upstream signal to activate proteoly sis in both cardiac and skeletal muscle. In addition, we tested the hypothesis that DOX induced activation of the calpain proteolytic system is essential for DOX induced damage to cardiac and ske letal myocytes via enhanced proteolysis and apoptosis. O ur fi ndings support this prediction and reveal that calpain does play a critical role in DOX induced myopathy in the heart and skeletal muscles and can contribute to muscle proteolysis and dysfunction Collectively, these findings indicate that DOX administrati on contributes to increases in mitochondrial ROS emission and increased calpain activity and that these signaling pathways play a required role in DOX induced myopathy A detailed discussion of these findings follows. DOX Administration Results in Cardiac a nd Skeletal Muscle Dysfunction Doxorubicin is a widely used and highly effective antitumor agent. However, its clinical efficacy is limited due to severe myotoxicity. In this regard, our data demonstrates that DOX administration rapidly results in a sever e cardiomyopathy.

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82 Specifically, treatment with DOX results in a thin ning of the septal and ventricular walls as well as changes to both fractional shortening and the myocardial performance index. In addition, our data demonstrates that DOX induced toxicity is not limited to the cardiac tissue. Specifically, our results revealed that diaphragm force production is significantly reduced as a result of DOX. This is significant because the diaphragm is the primary inspiratory muscle in all mammals [ 63 ] In addition, muscle fibers from the diaphragm, soleus and plantaris are all significa ntly reduced in size when compared to healthy control animals. This data demonstrates that DOX administration results in severe muscle dysfunction. However, our data also demonstrates that the DOX induced myopathy can be reduced through the administration of the innovative mitochondrial targeted peptide SS 31. In this regard, previous work from our laboratory has demonstrated that SS 31 administration is sufficient to protect against disuse muscle atrophy [ 62 ] [ 64 ] Importantly SS 31 administration protected both cardiac a nd skeletal muscle against DOX induced fiber atrophy and contractile dysfunction. Therefore, these results demonstrate that inhibiting mitochondrial ROS production is sufficient to protect the muscle from DOX induced side effects. Mitochondria are a Key So urc e of DOX Induced ROS Emission It is well established that DOX administration results in the increased formation of ROS disturbed redox signaling, and oxidative damage to cardiac and skeletal muscles [ 13 20 22 ] Specifically, DOX administration has been proposed to stimulate ROS generation by mitochondrial NADH dehydrogenase leading to the generation of a free radical cascade with potent oxidizing potentia l [ 8 9 ] Therefore, it is predicted that mitochondria play an important role in DOX induced ROS production. In reference to DOX induced mitochondrial dysfunction, o ur results clearly indicate that DOX

PAGE 83

83 administration results in a significant decrease in the mitochondrial RCR as well as an increase in mitochondrial ROS emission and that treatment with SS 31 protects both cardiac and ske letal muscles against DOX induced mitochondrial ROS production Furthermore, treatment of animals with SS 31 protected against DOX induced increases in oxidative damage to cardi ac and skeletal muscle proteins. In summary, these data demonstrate that mitochondrial ROS are the dominant cont ributor to DOX induced oxid ative damage in cardiac and skeletal muscles. Mitochondrial ROS Emission Promotes Protease Activation and Proteolysis Following DOX Administration Previous work indicates that DOX administration significantl y increase s the acti vity of the calpain, caspase 3 and ubiquitin proteasome system in cardiac and skeletal muscle [ 12 ] [ 13 ] In addition, increased cellular ROS production has been shown to be sufficient to activate a ll three proteolytic systems which results in the breakdown of ca rdiac and skeletal muscle proteins [ 13 ] [ 64 ] The current results indicate that DOX administration significantly increased the calpain and caspase 3 specific degrada tion products of alpha II spect rin in cardiac and skeletal muscles. However treatment with SS 31 protected cardiac and skeletal muscles against DOX induced activation of both calpain and caspase 3. Therefore, t he present experiments demonstrate that mitochondrial ROS emission is a requ irement for DOX induced activation of both calpain and caspase 3 in cardiac and skeletal muscles. In addition to the role of caspase 3 in protein degradation and muscle atrophy, DOX induced caspase 3 activation pro mote s DNA fragmentation and eventually apoptosis [ 65 ] Further, i t is established that DOX induces apoptosis in cells via a mitochondrial mediated pathways [ 66 ] Therefore, we determined the role that mitochondrial ROS

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84 emission plays in DOX induced apoptosi s in cardiac and skeletal muscles. DOX induced myonuclear apoptosis was determined by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) using a histochemical fluorescent detection kit. Our results reveal that DOX administration resulted in a significantly higher number of TUNEL positive nuclei in cardiac and skeletal muscle compared to control animals Importantly, treatment with SS 31 attenuated increases in TUNEL positive nuclei in cardiac and skeletal muscle following DOX administration. In summary, our data indicate that mitochondrial ROS emission is a key contributor to DOX induced apoptosis, leading to a loss of nuclei from cardiac and skeletal muscles. Finally, in addition to the importance of calpain and caspase 3, r ecent studi es have shown that treatment with DOX enhances proteolysis mediated by the ubiquitin proteasome system in cardiac and skeletal muscles [ 13 67 ] Since ubiquitin E3 ligases have been implicated in DOX induced pathologic conditions, increases in ubiquitin E3 ligase expression are important. Therefore, t o determine whether DOX induced mitochondrial ROS production is responsible for activation of the muscle specific E3 ligases in both cardiac and skeletal muscle we measured protein levels of two im portant muscle specific E3 ligases, MuRF 1 and atrogin 1 Our data indicate that DOX administration resulted in a significant increase in the levels of MuRF 1 and atrogin 1 in both cardiac and skeletal muscles. However, treatment with SS 31 significantly b lunted the DOX induced increases in MuRF 1 and atrogin 1 protein levels in cardiac and ske letal muscles. Collectively, these results reveal that mitochondrial ROS emission is a required upstream signal for DOX induced activation of key proteases and ubiqui tin ligases in cardiac and skeletal muscles.

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85 Calpain Activation Leads t o Muscle Dysfunction During DOX Administration As previously reported, administration of DOX results in contractile dysfunction and atrophy of both cardiac and skeletal muscles [ 68 69 ] In this regard, the protease calpain may play an important role in promoting dysfunction of both the cardiac and skeletal muscle s Previous reports have shown that calpain plays an important role in cardiac dysfunc tion in both heart failure and DOX induced myocardial dysfunction [ 16 70 ] Indeed it appears that calpain activa tion also plays an important role in DOX induced cardiomyopat hy. Specifically, inhibition of calpain attenuated the DOX induced a thinning of the septal walls (SWT) and posterior ventricular walls (PWT) during systole and provided partial protection against DOX induced decreased posterior wall shortening velocity a nd left ventricle systolic function Further, inhibition of calpain activatio n was sufficient to provide partial protection a gainst DOX induced decreased diaphragmatic s pecific force production at submaximal stimulation frequencies. Finally, while DOX results in an increase in muscle fiber atrophy of diaphragm, soleus and plantaris muscle fibers, ca lpain inhibition attenuates DOX induced muscle fiber atrophy in all three muscles. Together these re sults indicate that protection against DOX induced calpain activation provides protection against DOX induced atrophy and dysfunction in cardiac and skeletal muscles following DOX administration and that calpain plays an important role in regulating muscle function. Active Calpain Contributes t o DOX Induced Activation of Caspase 3 and Ubiquitin Proteasome System Calpains are calcium activated proteases that play an important role in skeletal muscle atrophy and weakness [ 62 ] [ 64 ] Specifically, calpain activation has been shown to induce muscle damage via many different pathways. For example, active calpain can

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86 cleave several structural proteins leading to the relea se of myofilaments, facilitating their subsequent degradation by the proteasome [ 71 ] Moreover activati on of calpain can also contribute to apoptosis through the cleavage of Bid, mediating cytochrome C release from the mitochondria leading to caspase 3 activation [ 27 ] Importantly, admi nistration of the calpain specific inhibitor SJA6017 was suffi ci ent to a ttenuate the increase in calpain activation in DOX treated animals. In addition to calpain mediated proteolysis, our data also reveals that pharmacological inhibition of calpain inhibi ted DOX induced caspase 3 activation as well as myonuclear apoptosis in cardiac and skeletal muscles. These findings reveal that calpain activation is required for caspase 3 induced apoptosis. Finally, as previously mentioned muscle specific E3 ubiquitin ligases play a critical role in ubiquitin proteasome system dependent muscle atrophy. Importantly, calpain appears to be an upstream regulator of both MuRF 1 and atrogin 1 protein expression. Indeed, inhibitio n of calpain act ivity in skeletal muscle prevents increases in both MuRF 1 and atrogin 1 protein levels in animals treated with DOX. Calpain Activation Upregulates Lysosomal Proteases Cathepsin L is a ubiquitously expres sed lysosomal pr otease that is charged with the rem oval o f both organelles and nonmyofib r illar cytosolic protein aggregates. Our prior work shows that cathepsin L gene expression is significantly increased as a result of DOX administration [ 65 ] In addition cathepsin L transcription may be regulated by calpain activity. O ur data are consistent with previous reports that DOX administration upregulates the expression of cathepsin L in cardiac and skeletal muscles [ 65 ] and pharmacological inhibition of calpain prevented this DOX induced increase in cathepsin L abundance in cardiac and skeletal muscles. Hence, these finding i ndicate that DOX

PAGE 87

87 induced calpain activation promotes the expression of lysosomal protease s in cardiac and skeletal muscles. Conclusions and Future Directions This study provides the first evidence regarding the role that mitochondrial ROS and calpain activation p lay in DOX induced myopathy in cardiac and skeletal muscles Specifically, o u r results demonstrate that mitochond ria are the dominant source of oxidant production in the cardiac and skeletal muscle during DOX administration. Indeed, mitochondrial ROS emiss ion is a required upstream signal to activate key proteases in response to DOX administration. Furthermore, our data also reveal s that calpain activation is a requirement for DOX induced myopathy in the heart and skeletal muscles, and ca lpain inhibition pr events DOX induced damage to cardiac and skeletal muscles. Collectively the current findings suggest that mitochondrial ROS emission and calpain activation contribute to the development of DOX induced myopathy in cardiac and skeletal muscles. These experi ments suggest that administration of a mitochondrial targeted antioxidant or inhibition of calpain m a y have therapeutic potential in protecting against DOX induced myopathy in the heart and skeletal muscles. Finally, u nderstanding the mechanisms responsible for DOX induced myopathy is important because the clinical application of DOX is greatly limited by its adverse effect s on several tissues and organs. Although the present experiments provide new and important information regarding the connecti on between DOX administration and cardiac and skeletal muscle myopathy, many unanswered questions remain. For example, the signaling pathway linking DOX induced mitochondrial ROS production to the activation of calpain remain unknown. Further, it is unclea r how calpain activation results in the activation of caspase 3. Moreover, it is undetermined if a regulatory cross

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88 talk exists between calpain and caspase 3 in cardiac and skeletal muscl es of animals treated with DOX Finally, the mechanism(s) responsible for calpain mediated expression of E3 ligases in cardiac and skeletal muscle remain unknown. Future experiments to address these unresolved issues will be important in determining the optimal therapeutic approach to the prevention of DOX induced cardiac a nd skeletal muscle myopathy.

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96 BIOGRAPHICAL SKETCH Kisuk Min was born in Seoul, Republic of Korea. He earned a Bachelor of Science degree in physical education from SungKyunKwan University in 1997. Following graduation, he has completed a military service as ROTC from 1997 to 1999. After military service, he began his graduate work at the Seoul National University in 2000. After his first semester, he had work at Samsung Medical Center as clinical exercise physiologist for 5 years. He had rehabilitated patients who have chronic diseases such as cardiovascul ar diseases, diabetes, and obesity etc. While he worked at the hospital, he received his Master of Science degree in 2004 in physical education, with a specialization in exercise physiology. Kisuk then began his doctoral program at the University of Florid a in Gainesville, Florida under the di rection of Dr. Scott Powers in the D epartment of Applied Physiology and Kinesiology. His research focused on mitochondrial oxidative stress responsible for cardiac and skeletal myopathy. He graduated in 2012 with a Doc tor of Philosophy degree in exercise physiology.