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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-05-31.

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Permanent Link: http://ufdc.ufl.edu/UFE0044075/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-05-31.
Physical Description: Book
Language: english
Creator: Senf, Sarah M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

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

Notes

Statement of Responsibility: by Sarah M Senf.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Judge, Andrew.
Local: Co-adviser: Dodd, Stephen L.
Electronic Access: INACCESSIBLE UNTIL 2014-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044075:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044075/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-05-31.
Physical Description: Book
Language: english
Creator: Senf, Sarah M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

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

Notes

Statement of Responsibility: by Sarah M Senf.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Judge, Andrew.
Local: Co-adviser: Dodd, Stephen L.
Electronic Access: INACCESSIBLE UNTIL 2014-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044075:00001


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1 THE R EQUIREMENT OF HSP70 IN THE REGULAT ION OF SKELETAL MUSC LE PLASTICITY: MUSCLE REGENERATION AND RECOVERY FOLLOWING INJURY By SARAH M SENF 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 Sarah M Senf

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3 To my parents, for their unwavering support encouragement and love

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4 ACKNOWLEDGMENTS I would like to thank my me ntor and committee chair, Dr. Andrew Judge for his guidance and support throughout my graduate studies at the University of Florida. I am forever grateful to him for sharing with me his passion for science and encouraging me to become an independent think er. I would not be where I am today had it not been for his friendship, and his sincere dedic ation to my growth and development as a research scientist. I would also like to thank my co chair, Dr. Stephen Dodd, for his continued support and mentorship thro ughout my graduate studies Further, I am grateful to my committee members, Drs. Scott P owers and Barry Byrne, for the roles they played in my education and their valuable input regarding my dissertation project. I also thank all of the former and current members of the Judge Lab for their much appreciated support and assistance in the lab. Special thanks go to Pooja Sandesara for her technical assistance in histology and to Travis Howard for his contributions in the completion of this project. Most importa ntly, I thank my parents, Stephen and Patricia Senf, and my sister, LeAnn Tinsley for their love and continued encouragement in my personal growth throughout the years. It is through their example that I strive to lead a life of love, passion and kindness Last, but not least, I would like to acknowledge those who shared a passing role in my life. I will forever be appreciative for their love, support and encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE ................................ ............... 11 Introduction ................................ ................................ ................................ ............. 11 Heat Shock Prote in 70 ................................ ................................ ............................ 12 Overview ................................ ................................ ................................ .......... 12 Hsp70 mediated Cellular Protection ................................ ................................ 13 Skele tal Muscle Plasticity ................................ ................................ ........................ 16 Regulation of Skeletal Muscle Mass ................................ ................................ 17 Muscle Regeneration and Recovery following Muscle Injury ............................ 21 Hsp70 Overexpression and Skeletal Muscle Plasticity ................................ ........... 23 Hypotheses and Aims of the Current study ................................ ............................. 26 2 MATERIALS AND METHODS ................................ ................................ ................ 28 Animals ................................ ................................ ................................ ................... 28 Cardiotoxin Model of Muscle Injury ................................ ................................ ......... 28 BrdU Labeling of Proliferating Cells ................................ ................................ ........ 28 Hind Limb Immobilization/Reloading Model of Muscle Injury ................................ .. 29 Histology ................................ ................................ ................................ ................. 29 Hematoxylin & Eosin (H&E) Staining ................................ ................................ 30 Von Kossa and Trichrome Staining ................................ ................................ .. 30 Immunohistochemistry ................................ ................................ ...................... 31 BrdU Immunostaining ................................ ................................ ....................... 31 Quantification of Muscle Necrosis, Muscle R egeneration and Fiber CSA ............... 32 RNA Isolation, cDNA Synthesis and qRT PCR ................................ ....................... 32 RT 2 Profiler PCR Arrays ................................ ................................ ......................... 33 Western Blot Analyses ................................ ................................ ............................ 3 4 In vivo Muscle Transfection and Rescue Experiments ................................ .......... 34 Statistical Analyses ................................ ................................ ................................ 35 3 RESULTS AND DISCUSSION ................................ ................................ ............... 36 Genetic Deletion of Hsp70 Impairs Skeletal Muscle Fiber Growth .......................... 36 Hsp70 / Mice have Impaired Muscle Regeneration ................................ ................ 38

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6 Persistent Muscle Inflammation in Hsp70 / Mice following Injury ............................ 40 Calcification of Regenerating Muscle from Hsp70 / Mice following Injury ............... 41 Muscles from Hsp70 / Mice Show Altered Gene Expression Profile 4 Days Post Cardio toxin Injury ................................ ................................ ................................ 42 Muscles from Hsp70 / Mice do not Show Deficits in Cellular Proliferation following Injury ................................ ................................ ................................ ..... 44 Muscles from Hsp70 / Show Impaired Muscle Recovery up to 6 weeks Post Injury ................................ ................................ ................................ .................... 46 Muscle Regrowth and Regeneration following Reloading induced Injury is Impaired in Hsp70 / Mice ................................ ................................ ..................... 47 Restoration of Hsp70 in Skeletal Muscle Restores Regenerative Deficits in Hsp70 / Mice ................................ ................................ ................................ ....... 52 4 CONCLUSIONS AND FUTURE DIRECTIONS ................................ ...................... 72 LIST OF REFERENCES ................................ ................................ ............................... 76 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 85

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7 LIST OF FIGURES Figure page 3 1 Confirmation of Hsp70 mRNA and protein knockout in Hsp70 / mice ................ 56 3 2 Lifelong Hsp70 knockout does not alter skeletal muscle development and morphology in 4 w eek old mice ................................ ................................ .......... 57 3 3 Decreased fiber cross sectional area in soleus muscles of 12 week old Hsp70 / mice ................................ ................................ ................................ ...... 58 3 4 Abnormal morpholo gy in Hsp70 / TA muscles 4 days po st cardiotoxin injury .... 59 3 5 Muscles from Hsp70 / mice show deficits in regeneration 16 days post cardiotoxin injury ................................ ................................ ................................ 60 3 6 Muscles from Hsp70 / mice show increased numbers of CD68 positive macrophages 16 days post cardiotoxin injury ................................ .................... 61 3 7 Hsp70 / mice develop calcifications in mu scl es following cardiotoxin injury ....... 62 3 8 Hsp70 / mice show altered gene expression 4 days post cardiotoxin injury. ...... 63 3 9 Meas urement of BrdU positive proliferating cells in muscles from WT and Hsp70 / mice following cardiotoxin injury. ................................ .......................... 64 3 10 Muscles from Hsp70 / mice show increased collagen deposition post cardi otoxin injury ................................ ................................ ................................ 65 3 11 Regenerating muscles from Hsp70 / mice fail to recover up to 6 weeks post cardiotoxin injury ................................ ................................ ................................ 66 3 12 Musc les from Hsp70 / mice show impaired regeneration and recovery following reloading injury. ................................ ................................ ................... 67 3 13 Inflammation and regeneration in soleus muscles of WT and Hsp70 / mice following reloading i njury. ................................ ................................ ................... 68 3 14 Altered gene expression in muscles of Hsp70 / mice following reloading injury ................................ ................................ ................................ ................... 69 3 15 Restoration of Hsp70 in mu scle fibers 4 days prior to cardiotoxin inj ury rescues deficits in regeneration and recovery in Hsp70 / mice. ......................... 70 3 16 Restoration of Hsp70 in regenerating muscle fibers of Hsp70 / mice 4 day s following cardiotoxin in jury visually increases regenerating fiber size ................ 71

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8 LIST OF ABBREVIATION S BrdU b romodioxyuridine CD68 c luster of di fferentiation 68 CSA c ross sectional area CTX c ardiotoxin eMHC embryonic myosin h eavy c hain FoxO f orkhead boxO H&E h ematoxylin and eosin Hsps h eat shock proteins IGF 1 i nsulin like g rowth f actor 1 IKK inhibitory of B k inase iNOS Inducible Nitric Oxide Synthase MuRF1 m uscle RING finger 1 NF B nuclear f actor B PBS p hosphate buffered s aline PCR polymerase chain reaction PI3K p hosphatidylinositol 3 kinase TA t ibialis anterior

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9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Deg ree of Doctor of Philosophy THE R EQUIREMENT OF HSP70 IN THE REGULATION OF SKE LETAL MUSCLE PLASTICITY: MUSCLE REGENERATION AND RECOVERY FOLLOWING INJURY By Sarah M Senf M ay 2012 Chair: Andrew R. Judge Cochair: Stephen L. Dodd Major: Health and Human P erformance The inducible 70 kDa Heat shock protein (Hsp70) is expressed in virtually all cell types and as a protein chaperone, plays a significant role in protecting against cellular damage and distress. Hsp70 protein levels are progressively decreased in skeletal muscle during aging, which is associated with the age related decline in skeletal muscle mass function and regenerative potential Importantly Hsp70 is also decreased during skeletal muscle disuse, and restoration of its levels is sufficient to prevent muscle fiber atrophy. Furthermore Hsp70 overexpression has also been shown to promote both muscle regeneration and functional recovery following injury, further demonstrating the breadth of cellular protection provided by Hsp70 to skeletal musc le. While the protective nature of Hsp70 demonstrated in these studies may indeed be related to its ubiquitous role as a general protein chaperone, we hypothesize that Hsp70 is in fact an indispensible protein in skeletal muscle necessary for normal skelet al muscle plasticity. In this regard, we hypothesize that a decrease in Hsp70 protein levels alone, as occurs during both muscle disuse and during the aging process, is sufficient to both alter skeletal muscle homeostasis under normal conditions and impede the normal

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10 regenerative and recovery process that occurs following injury and modified muscle use. The current study tested these hypotheses through the use of genetically modified mice which lack Hsp70 (Hsp70 / mice) We found that muscles from Hsp70 / mice had significantly smaller skeletal muscle fibers than those of WT mice under normal physiological conditions and were severely impaired in their ability to recover following injury due to cardiotoxin or modified muscle use Specifically, Hsp70 / mi ce displayed increased numbers of necrotic muscle fibers, increased mRNA levels of pro inflammatory cytokines and calcium activated proteases, increased numbers of CD68+ macrophages and deficits in regenerating fiber size that pers isted up to 6 weeks post injury. Interestingly, injured muscles from Hsp70 / mice also developed calcifications and significant fibrosis, characteristic of dystrophic muscle phenotypes Collectively, findings from this study demonstrate that Hsp70 is necessary for normal skeletal muscle fiber size and is further required for successful muscle regeneration and recovery following muscle injury.

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11 CHAPTER 1 I NTRODUCTION AND REVI EW OF LITERATURE Introduction Heat shock protein 70 (Hsp70) expression is induced in response to a va riety of physiological insults (55, 66) and is believed to be a significant player in protecting skeletal muscle from cellular damage and distress. The mechanistic nature of Hsp70 mediated cellular protection is be lieved to be related to both its role as a protein chaperone in regulating protein turnover (5, 27) and its ability to modulate cell signaling pathways As a chaperone, Hsp70 plays a dual role in preventing global p rotein denaturation and degradation following mild to moderate protein damage (5, 66) while promoting the degradation & clearance of irreversibly damaged proteins. In terms of its role in regulating cell signaling events, Hsp70 overexpression studies have demonstrated that Hsp70 can regulate the activities of various signaling proteins and transcription factors, including j un N terminal kinase ( JN K ) (10) p38 (20) n uclear factor B ( NF B) (10, 20, 71, 80) and f orkhead boxO (FoxO) (79, 80) Importantly, in skeletal muscle, these signaling proteins are involved in the regulation of various muscle remo deling processes including myogenesis and muscle growth (3) regeneration following injury (58) and muscle atrophy (6, 33, 35, 95) Perhaps more importantly in these studies Hsp70 overexpression ultimately protected against disruptions in muscle homeostasis in response to varying physiological and pathophysiological stimuli. Protection against muscle atrophy (80) and muscle damage (4) and promotion of regenerative processes were all achieved through muscle sp ecific Hsp70 overexpression studies, highlighting a seemingly critical role for Hsp70 in regulating skeletal muscle plasticity Interestingly, Hsp70 is down regulated during the aging

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12 process, in which reductions in muscle mass and function and impaired re generation following injury are all a significant problem. However, it is currently unknown whether a decrease in Hsp70 alone can cause disruptions in these cellular processes which ultimately regulate skeletal muscle mass and function Determining the rel ative phenotype of skeletal muscles which lack Hsp70, and further determining whether these muscles can successfully regenerate and recover following injury would provide critical information not currently known on the biological requirement of Hsp70 in sk eletal muscle remodeling Importantly knowledge gained from these studies may extend the clinical potential for Hsp70 in the treatment of a variety of skeletal muscle disorders and w ould ideally enhance the momentum toward the development of Hsp70 target ed therapeutics that can rapidly translate to future clinical studies Heat Shock Protein 70 Overview Heat shock proteins (Hsps) are a group of proteins which act as cellular chaperones, assisting in the folding of nascent polypeptides and the re folding of damaged proteins. Hsps are generally separated into two groups based on their molecular weight, and includes the high molecular weight Hsps and the low molecular weight Hsps. The Hsp70 family belongs to the ATP dependent, high molecular weight group of Hsps (87) along with the Hsp90 and Hsp60 families. Of the cytoplasmic Hsp70 family members the two most studied members are Hsc70, which is constitutively expressed, and the stress inducible Hsp70. As cellular chape rones Hsc70/Hsp70 function to help fold nascent proteins and re fold damaged proteins into their correct tertiary structures (16) Importantly however, when proteins are irreversibly damaged or even designated for protein degradation, Hsc70/Hsp70 can help shuttle these proteins

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13 to the proteasome for their subsequent degradation (39) This involvement of Hsc70/Hsp70 in protein degradation plays an important role in preventing protein aggregation and promoting cellular homeostasis The relative co chaperones which interact with Hsc70/Hsp70 appear to play important roles in the fate o f Hsc70/Hsp70 clie nt protein s The co chaperones CHIP (c terminus of Hsp70/Hsc70 interacting protein) and BAG 1 assist in Hsc70/Hsp70 mediated degradation of client proteins (11, 59, 88) CHIP is a ubiquitin liga se that when in complex with Hsc 70/Hs p 70, can ubiquitinate client proteins allowing for their subsequent degradation through the ubiquitin dependent proteasome system (59) BAG 1 interacts with the 20S core and 19S subunit of the proteasome and facilitates the rel ease of Hsp70 client substrates to the proteasome (45) In contrast, the co chaperones Hip and Hop assist Hsp70 in the folding and r e folding of protein substrates (18, 30) Therefore, dictated by which co chaperone proteins Hsc70/Hsp70 interacts with, Hsc70/Hsp70 can either promote pro tein folding and stability or promote protein degradation. Since Hsc70 and Hsp70 are believe d to be functionally similar due to their high homology, insight into the relative importance of these separate gene products can be gained from the conditions in which they are differentially regulated. In this body of work the specific focus will be on t he role of the stress inducible Hsp70 in skeletal muscle Hsp70 is altered in skeletal muscle in response to numerous physiological and environmental stimuli, which suggests a role for this protein in the subsequent skeletal muscle remodeling processes whi ch simultaneously occur in response to these stimuli. Hsp70 mediated Cellular Protection Various environmental stimuli, including free radicals, oxygen or nutrient deprivation and mechanical injury may result in the loss of protein and cellular integrity

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14 r esulting in alterations in cellular homeostasis (21) Elevation of Hsp70 during these stress conditions promotes cell survival and helps return cells to their normal homeostatic state (44, 63) which is critical to maintaining cellular function The protective nature of H s p70 ha s been linked to both its general chaperone function, as well its ability to regulate various kinases involved in cellular signaling including the stress kinases JNK and p38 (20) and the Inhibitor of B kinase (IKK ) complex (71) Regardless of the precise mechanism of protection, it can be co ncluded from these studies that induction of Hsp70 is a highly protective mechanism involved in cellular stress tolerance. However, as cells age and are exposed to continu ous cycles of cellular stress, a loss of cellular integrity is sometimes unavoidable. Fortunately, many cell types undergo numerous cycles of proliferation throughout their lifetime such that older and/ or irreversibly damaged cells that have severely declined in their cellular integrity can undergo programmed cell death, leaving behind su ffici ent levels of healthier or cells to carry out tissue function Some tissues like the skin, replace cells every few weeks or months In contrast, some tissues are comprised of cells that are post mitotic, such as neuronal cells in the brain an d skeletal muscle fibers (cells) in skeletal muscle. These post mitotic cells do not undergo further cellular proliferation, and may live for a lifetime Therefore, maintenance of protein and cellular integrity in post mitotic cells is especially critical for cellular and tissue function. The importance of Hsp70 in promoting cellular integrity in post mitotic neuron al cells especially in aged populations, has been shown in numerous studies. Hoshino et al recently demonstrated the pr eventative effect of neu ronal specific Hsp70 transgenic expression in a rodent model of

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15 disease (AD) (31) Indeed, the build up and aggregation of beta amyloid protein, which is involved in the pathogenesis of AD was prevented by Hsp70 overexpression, as were the behavioral manifestations of AD such as memory loss (31) Hsp70 overexpression also interfere s with the patho logy of Amyotrophic Lateral Sclerosis (22) (99) additional diseases whose patho logy is characterized by defects in protein quality control that typically begin to occur later in life. As previously mentioned, skeletal muscle fibers are also post mitotic cells A lthough multi nucleated skeletal muscle fibers do undergo turnover of cellular nuclei through apoptosis of existing fiber nuclei and replacement of these n uclei through the fusion of activated muscle satellite cells (7) individual muscle fibers may remain for a lifetime. Th erefore, similar to neuronal cells in the maintenance of brain and motor neuron function, preserving protein integrity and cellular homeostasis in skeletal muscle fibers is especially critical to the maintenance of skeletal muscle function Due to the role s of Hsp70 in regulating cellular homeostasis Hsp70 has for many years been speculated to play an important role in skeletal muscle to promote homeostasis and overall muscle health (42) In this regard, Hsp70 protein expression is induced in skeletal muscle in response to exercise and muscle activity, which is believed to protect the muscle from alterations in protein and structural integr ity that may occur during these conditions due to elevations in muscle temperature, mechanical st rain, oxidative stress and changes in pH, to name just a few. Importantly, Hsp70 protein expression declines with age, and the relative ability to increase Hsp 70 in skeletal muscle in response to exercise and muscle activity is also blunted with age (51, 96) This decrease in Hsp70 is associated with the age related reductions in skeletal muscle

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16 mass, function and regene rative potential as increasing the levels of Hsp70 in aged mice through transgenic expression results in increased function under normal conditions and enhanced recovery following exercise induced muscle damage when compared to aged wildtype mice (50) This evidence which shows that Hsp70 overexpression can protect against these age related deficits in skeletal muscle plasticity further suppo rts the importance of Hsp70 in regulating cellular homeostasis and function Therefore, the speculation that decreased levels of Hsp 70 alone may contribute to these age related deficits in skeletal muscle is not without warrant (51) Prior to discussing further the evidence which supports Hsp70 in regulating skeletal muscle plasticity, the next chapt er will provide an introdu ction to skeletal muscle and it s extraordinary a bilities to adapt to its environment and recover following even the most severe cases of cellular stress and damage Based on the functional properties of Hsp70 discussed above, hope fully one will appreciate the potential roles that Hs p70 may specifically p lay in regulating the skeletal muscle processes that provide muscle its plastic nature. Skeletal Muscle Plasticity In biology, plasticity can be defined as the ability to adapt to e nvironmental and/or functional demands, which may be reflected by changes in both structural and/or functional phenotype In ske letal muscle, plasticity may be reflected by changes in fiber cross sectional area and leng th as well as fiber type shifts, whic h may occur following modified muscle activity (76) However, perhaps the most extraordinary plastic feature of skeletal muscle is its ability to self renew. This ability to self r enew or regenerate is provided by skeletal muscle satellite cells. Satellite cells are specialized muscle stem cells that are located adjacent to muscle fibers, underneath the basement membrane.

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17 Activation and f usion of satellite cells into muscle fibers a llow for post natal muscle growth and maturation, as well as muscle re growth, repair and even complete regeneration following injury (7) Interruptions in any of these skeletal muscle processes can result in significant impairments in skeletal muscle function. Indeed, n umerous skeletal muscle disorders including the genetic muscular dystrophies (97) and inflammatory myopathies (48) are cha racterized by significant impairment s in skeletal muscle function due to interruptions in these critical skeletal muscle processes. Even various physiological conditions such as prolonged muscle inactivity and aging (47) and pathophysiological conditions such as cancer and s epsis (56) can result in impaired muscle function due to the downstream consequences of these conditions on these cellular processes in skeletal muscle Importantly, these deficits in skeletal muscle function often have devastating consequences to patient health and even survival (56, 97) Therefore, u nderstanding the detailed mechanisms underlying skeletal muscle plasticity is the foundation for future clinical treatments wh ich aim to exploit the plastic nature of skeletal muscle to treat and ideally cure these devastating conditions. As the curren t work will focus on the importance of Hsp70 in skeletal muscle plasticity a more detailed introduction to the skeletal muscle pr ocesses that give muscle its plastic nature will be discussed, focusing specifically on the regulation of skeletal muscle mass (atrophy and hypertrophy) as well as muscle re growth and regeneration following injury Regulation of Skeletal Muscle Mass T he maintenance of skeletal musc l e mass occurs through balancing protein degradation and protein synthesis, which is mediated through both circulating systemic and local growth and growth inhibitory factors (e.g. Insulin like Growth Factor 1 [ IGF 1 ]

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18 and myosta tin) and the mechanical load placed on the muscle (23, 34, 74) In response to increased skeletal muscle load ing or administration of growth factors, muscle fibers can hypertrophy or increase in mass and fiber cross sectional area (24) On the other hand skeletal muscle c an decrease in size (atrophy) in response to decreased muscle loading, in response to certain circulating factors such as inflammatory cytokines that are elevated during various disease conditions (2) or in response to deficits in normal growth signalin g pathways (34) Skeletal muscle atrophy occurs in response to a variety of both physiological and pathophysiological stimuli, and contributes to profound losses of muscle mass and whole body strength (34) Among the physiological stimuli which trigger the loss of muscle mass is muscle disuse, prolonged muscle inactivity that results in reduced mechanical loading or tension on the muscle (26, 90) Atrophy resulting from cast immobilization of a limb, bed rest, or a general inactive lifestyle each fall under the category of disuse muscle atrophy, as does atrophy resulting from interruptions in neural input to the muscle, such that oc curs during functional denervation in elderly patients (46) In contrast, cachectic muscle wasting is a form of pathophysiological wasting that oft en accompanies chronic diseases such as cancer (2) and chronic heart failure, and more acute conditions such as sepsis (28) Although patients affected by these conditions may suffer from some level of disuse wasting as a consequence of inactivity due to their disease, cachectic wasting (cachexia) is believed to predom inately result from increased levels of systemic circulating factors such as inflammatory cytokines that are elevated by the host immune system in response to the disease (93) In addition to immune derived factors, tumor secreted factors in cancer patients, and

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19 blood borne lipopolysaccharide from the bacterial infection in septic patients, are also implicated in the pathogenesis of muscle wasting (93) Therefore, although there are multiple distinct triggers of muscle wasting, in the clinical population, muscle wasting is often a complex condition that may involve numerous underlying triggers. Importantly, t he loss of muscle mass not only effects muscle strength and function, it also increases the risk for metabolic disorders such as diabetes contributes to prolonged and/or impaired recover y following hospital stays and in the most severe cases, contribu tes to increased mortality (56, 94) Therefore, improving our understanding of how skeletal muscle atrophy is regulated is clinically significant for not only combating or alleviating the muscle atrophy itself, but enh ancing patient health and survival. Despite the numerous upstream triggers of skeletal muscle atrophy, the loss of skeletal muscle mass and function results from the preferential loss of skeletal muscle contractile proteins (19, 83) During normal physiological conditions, skeletal muscle contractile proteins are degraded at a similar rate in which they are synthesized, which results in an overall maintenance of healthy muscle proteins and maintenance of muscle mass. In response to atrophic stimuli, protein degradation through the ubiquitin proteasome system is elevated significantly (89) while protein synthesis coordinately declines, resulting in a net decrease in skeletal muscle contractile protein. Since these changes in protein turnover ultimately dictate whether a muscle will atrophy, hypertro phy or remain a certain fiber size understanding how these cellular processes are regulated in skeletal muscle has become a major research focus in recent years. Numerous proteins and signaling pathways have been identified to regulate skeletal mass thro ugh modulating the balance between protein synthesis and protein

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2 0 degradation (24) Growth signaling through Akt both stimulates protein synthesis through activating mTOR signaling and inhibits protein degradation pathways through phosphorylating and inhibiting the Fo rkhead boxO (FoxO) transcription factors (75, 85) Fox O transcription factors are activated during atrophic conditions including muscle disuse (75, 80) cancer and sepsis (82) and their activation is required for normal muscle wasting (72, 79) Fox O transcription factors cause muscle atrophy via the ir transcription al regulation of multiple genes involved in proteolysis including genes invol ved in the ubiquitin proteasome system (75) FoxO also regulates genes involved in autophagy (49) which is an important cellular process involved in the basal turnover of ce llular orga nelles during normal physiological conditions that is accelerated during certain conditions of muscle atrophy (49) Importantly, our lab group recently demonstrated that decreased levels of Fox O signaling under normal physiological conditions can lead to skeletal muscle hypertrophy (72) Although this is likely related in part to decreased levels of basal protein turnover, this hypertrophy also required protein synthesis and involved both satellite cell proliferation and fusion and decreased myostatin signaling. Th erefore, it appears that Fox O regulates not only proteolytic pathways during atrophic conditions but also plays an important role in preventing muscle fiber hypertrophy during normal physiological conditions. There is also strong evidence that NF B signaling is involved in the regulation of skeletal muscle mass (68) Indeed NF B transcriptional activity is increased during atrophic conditions (6, 33, 67) and inhibition of NF B via genetic manipulation of various proteins in the NF B signaling pathway, including p50, Bcl3, I B IKK a nd IKK are all sufficient to inhibit the normal atrophy program (6, 32, 35, 58, 95) While the

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21 precise role in which NF B activation contributes to muscle loss during atrophy conditions is still not well defined, the atrophy gene MuRF1 is a known NF B target gene (6) While n umerous other signaling pathways and proteins have also been identified to regulate skeletal muscle mass during normal physiological and pathophysiolog ical conditions (74) due to the scope of the current study, these signaling pathways will not be discussed. Mu scle Regeneration and Recovery f ollowing Muscle Injury Skeletal muscle not on ly has the ability to change the size of individual muscle fibers ( atrophy/hypertrophy ), it also has a remarkable capacity to repair and even replace damaged muscle fibers following injury (7) This capability of skeletal muscle to muscle stem cell known as a satellite cell. Following muscle i njury (mechanical or chemical), factors released by the injured muscle and by resident and invading inflammatory cells stimulate these normally quiescent satellite cells to activate, proliferate, and eventually differentiate and fuse with either existing m uscle fibers or to other myogenic cells to generate new myotubes (7, 91) Impairments in any one of these coordinated events which lead to muscle regeneration profoundly limit the ability of skeletal muscle to recov er following muscle injury (7, 91) Muscle injury may occur in response to a v ariety of physiological insults including crush injury but more commonly, following mechan ical strain on the muscle due to eccentric o r damaging muscle contractions, and during muscle reloading following prolonged period s of muscle unloading and disuse (7, 47) Although mechanical injury is the primary physiological cause of muscle in jury, chemical s (reactive oxygen species,

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22 reactive metals, and/or toxin s ) can also induce muscle inju ry Regardless of the nature of acute muscle damage, the general time course in which specific myeloid cell populations accumulate in the muscle and myogenesis proceeds is similar, and well defined (7, 91) Indeed follow ing the initial bout of injury the innate im mune response is activated, leading to the infiltration of neutrophils, which may peak in concentration between 6 and 2 4 hours post injury (7, 91) Neutrophils play an important ro le in the release of myeloperoxidase, which induces m uscle membrane damage and amplification of muscle injury (60, 62) Importantly, soon after the invasion of neutrophils, phagocytic pro inflammatory (M1) macrophages begin to invade and peak in concentration 24 48 hours post injury, followed by a sharp decline in numbers thereafter (91) These pro inflammatory macrophages help remove necrotic and damaged muscle fibers and debris and also release both growth factors and pro inflammatory cytokines that are involved in stimulating satellite cell activation and proliferation (86, 91) However, overproduction and/or sustained release of cytokines by these macrophages can also inhibit regenerative processes. T herefore soon after the peak in the pro inflammatory M1 macrophages, th ere is shift in these macrophages toward an anti inflammatory (M2) phenotype (91) Th is shift in macrophage phenotype towards an anti inflammatory phenotype c orresponds with the shift from the proliferative phase of muscle rege neratio n to the differentiation phase. Indeed, th ese M2 macrophages secrete anti inflammatory cytokines that are involved in deactivating the pro inflammatory immune response and promot ing muscle differentiation, repair and growth of regenerating fibers (91, 92) Dep ending on the severity of the damaging stimuli, restoration of normal fiber architecture usually occurs around 10 days following

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23 muscle injury followed by restoration of mature fiber cross sectional area by 3 4 weeks post injury (7) Hsp70 Overexpression and Skeletal Muscle Plasticity Hsp70 has long been speculated to pla y a role in protecting skeletal muscle under stressful stimuli as discussed earlier Hsp70 is elevated in skeletal muscle following exercise (29, 52, 57) as well as during periods of muscle re growth following mus cle disuse (78) In contrast, Hsp70 is decreased during periods of muscle inactivity (9, 40, 80) when muscles undergo remodeling to reduce their size according to the n ew reduced mechanical load on the muscle. Given this association of elevated Hsp70 levels during periods of muscle activity, growth and regeneration and decreased levels during periods of muscle inactivity and muscle atrophy, the idea that Hsp70 may posit ively regulate muscle mass is not without warrant In support of this notion, previous studies using heat stress to increase heat sh ock proteins in skeletal muscle have shown that heat stress can provide some level of protection again st disuse muscle atrop hy (77) and can promote muscle regrowth (78) Similarly, heat stress prior to or followin g muscle injury due to bupivacaine (64) or cardiotoxin (36) promoted satellite cell activation and earlier recovery of regenerating skeletal muscle fiber size, further indicating a role for heat shock proteins in regulating muscle regeneration However, as heating may alter the activation of multiple cellular signaling pathways and differentially regulate the protein expression of many proteins in add ition to Hsp70, these heating studies are only suggestive evidence of the role that Hsps and Hsp70 may play in regulating skeletal muscle plasticity More direct evidence to specifically implicate Hsp70 in the regulation of skeletal mass and function c ome s from studies which have genetically overe xpressed Hsp70.

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24 Muscles from skeletal muscle spe cific Hsp70 overexpresso r mice recover quicker, both morphologically and functionally, following muscle damage due to eccentric lengthening contractions and are also protected from the age related loss in specific muscle force (50) Similarly, muscles from Hsp70 transgenics show less damage following cry olesioning inju ry compared to WT as indicated by less inflammatory cell infiltration and numbers of necr otic fibers one day post injury (53) M uscles from Hsp70 transgenics in this study also showed lower levels of satellite cell activation and proliferation This finding was speculated to be related to the decreased muscle damage in the Hsp70 transgenics which would thereby result in a decreased need for satellite cell mediated repair of muscle fibers An addition al study using plasmid mediated overexpression of Hsp70 following freeze injury further demonstrated that Hsp70 enhances regenerating muscle fiber size (54) Together these studies provide strong evidence that Hsp70 indeed is sufficient to both protect skeletal muscle from damage in the event of injury, and promote muscle regeneration and recovery. Importantly, Hsp70 protein levels are increased in muscle biopsies from patients diagnosed with However, whether Hsp70 i s required for muscle regeneration and recovery in the event of muscle injury is not currently known. In addition to a role for Hsp70 in muscle recovery following injury, Hsp70 has also been shown to protect against skeletal muscle atrophy during muscle d isuse (80) Indeed, Hsp70 l evels are significantly downregulated during muscle disuse, and p lasmid mediated restoration of Hsp70 in skeletal muscle prevent s greate r than 75% of the fiber atrophy (80) Findings from this study indicate that a decrease in endogenous Hsp70 during disuse may be a n important event allowing the atrop hy program to

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25 proceed Since Hsp70 protein is also decreased in aged skeletal muscle (14, 51) it may also be speculate d that the age related loss of muscle mass may similarly be related to the dec reased levels of Hsp70. However, whether a decrease in Hsp70 protein alone is sufficient to decrease skeletal muscle fiber size has not been established. Important additional information gained from experiments performed in these studies in skeletal muscl e and in studies fr om other cell types, indicate that Hsp70 can modulate specific int racellular signaling pathways. Indeed, Hsp70 overexpression represses NF B signaling in skeletal muscle (80) a pathway whose activation is well established to be necessary for muscle fiber atrophy d uring various atrophic conditions (6, 32, 35, 95) Signaling through NF B also limit s skeletal muscle growth (3, 68) and impair s muscle regeneration (58) Additionally, Hsp70 overexpression also represses F oxO signaling in skeletal muscle (79, 80) another pathway known to be required for muscle fiber atrophy (72, 75, 79) and which also limits skeletal muscle fiber hypertrophy under normal conditions (72, 73) Hsp70 also decrease s the activity of JNK in skeletal muscle a stress kinase whose activity in skeletal mus cle is linked to inflammatory signaling and insulin resistance (10) Indeed, muscle specific Hsp70 transgenics are protected from diet and obesity induced glucose intolerance and insulin resistance, which was tight ly as sociated with repression of JNK and IKK (10) Therefore, the ability of Hsp70 to regulate these critical cell signaling proteins in skeletal muscle may explain, in part, the findings that up regulation of Hsp70 can preserve muscle mass under catabolic conditions, and enhance muscle recovery following injury. However, no studies currently exist to demonstrate the physiological requirement of Hsp70 for any skeletal muscle adaptation. Therefore it is currently unkn own whether Hsp70 is indeed

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26 indispensible for normal skeletal muscle mass and phenotype during normal conditions and whether Hsp70 is further required for the coordinated cellular events that occur in skeletal muscle following injury that allow for normal muscle re growth and recovery. Hypotheses and Aims of the Current study N umerous studies from indepen dent lab groups have provided clear evidence to suggest a critical role for Hsp70 in regulating skeletal muscle plasticity. To summarize, these studies sh ow th at, 1) skeletal muscle expression of Hsp70 is decreased progressively with age, and is associated with the age related loss of muscle mass and regenerative potential ; 2 ) Hsp70 is decreased during periods of muscle disuse, and restoration of its levels can prevent fiber atrophy; 3 ) Hsp70 overexpression enhances regenerating fiber size and functional recovery following muscle injury and; 4 ) Hsp70 represses the activation of multiple signaling pathways in skeletal muscle which negatively regulate skeletal muscle mass and regenerative potential. Despite this evidence and the wide speculation that Hsp70 is central to the ability of skeletal muscle to withstand and respond to stress there are currently no studies that have directly considered the requirement of Hsp70 in any type of skeletal muscle adaptation Therefore, the aim of the current study was to test the central hypothesis that skeletal muscle expression of Hsp70 is indispensible for normal skeletal muscle phenotype under normal conditions, and is f urther necessary for the normal recovery process following muscle injury. The current study tested the above hypothes i s through the use of wild type and genetically modified mice that lack Hsp70 (Hsp70 / mice). Skeletal muscle f iber type distribution s f iber cross sectional area, total fiber numbers and general morphology w ere assessed in wild type (WT) and Hsp70 / mice under normal physiological

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27 conditions, to determine whether Hsp70 is necessary for normal muscle phenotype. To further determine whether Hsp70 is necessary for normal muscle regeneration and recovery following muscle injury we utilized tw o distinct models that are well used in the study of muscle injury and recovery, cardiotoxin injury and modified muscle use. Both biochemical and histolog ical measurements were used at various time points post injury to assess the relative levels of muscle damage, inflammation and regenerative capabilitie s of muscles from WT and Hsp70 / mice. Lastly, rescue experiments were performed via the electroporatio n of Hsp70 E GFP or E GFP specifically in skeletal muscles of Hsp70 / mice either 4 days prior to or 4 days following muscle injury to determine whether any deficits in skeletal muscle regeneration and recovery were indeed due to the lack of muscle derived Hsp70 at these time points.

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28 CHAPTER 2 MATERIALS AND METHOD S Animals All experimental animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida. B6 129S7 Hspa1a/Hspa1b tm mice (hereafter referred to as H sp70 / mice) were originally generated at the US Environmental Protection Agency (13) and were purchased from the Mutant Mouse Regional Resource Center (MMRRC) at the University of California, Davis Th e ir wild typ e counterparts B6129SF2/J mice (hereafter referred to as WT mice) were purchased f rom Jackson Laboratories (Bar Harbor, ME). All animals were housed in a sterile, pathogen free temperature con trolled facility on a normal 12 hr light/dark cycle and s tand ard diet and water were provided ad libitum. Cardiotoxin Model of Muscle Injury To induce muscle degeneration/regeneration, 100 l of 10 M cardiotoxin (Calbiochem) dissolved in PBS was injected into either the right or left tibialis anterior (TA) muscle of 6 8 week old WT or Hsp70 / mice. Cardiotoxin is a protein kinase C specific inhibitor and its injection into skeletal muscle induces muscle depolarization, contraction and subsequent fiber necrosis in greater than 9 0 % of muscle fibers (100) Animals were sacrificed with a lethal dose of pentobarbital and muscles were harvested 1, 4, 6 1 6, 28 or 42 days post cardiotoxin injection The uninjured, contralateral TA muscles from each animal served as controls. BrdU Labeling of Proliferating Cells T o measure cellular pr oliferation in TA musc les in response to cardiotoxin injury, mice were inj ected intraperitoneal ( IP ) daily for 6 days with 50mg/kg Br omodeoxyuridine

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29 (Br dU ) dissolved in sterile saline beginning 6 hours prior to cardiotoxin injury BrdU is a thymidine analogue that is incorporated into DNA during DNA replication, which occurs dur ing cellular proliferation. Since skeletal muscle satellite cells proliferate extensively following muscle injury due to cardiotoxin, staining for BrdU intensity and localization in skeletal muscle fibers in muscle cross sections can provide important info rmation on satellite cell proliferative capacity and regeneration involving satellite cell activation. Injured and contralateral non injured TA muscles were surgically removed 6 days post cardiotoxin injury, embedded in OCT freezing medium and frozen in li quid nitrogen cooled isopentane prior to storage at 80C. Hind L imb Immobilization/R eloading M odel of Muscle Injury Muscle reloading injury was induced via bilateral hind limb cast immobilization with ankles in the plantar flexed position for 10 days as d escribed previously (ref), followed by cast removal and reambulation for either 3 or 10 days. Briefly, a layer of protective padding was applied to both hind limbs and the lower abdominal area (Medipore). Extra fast drying plaster (3M) was then wetted and applied over the protective padding and allowed to dry. A thin layer of casting tape (3M) was then applied over the plaster to prevent mice from chewing through the plaster casts S keletal muscle unloading/ reloading injury in the soleus has been demonstra ted by numerous lab groups following both hind limb immobilization as performed in the current study (17) as well as following hind limb suspension (61, 91) Histology Skeletal muscles from Hsp70 / or WT mice to be used for histology were removed, embedded in OTC and immedi ately frozen in isopentane cooled in liquid nitrogen prior to storage at 80C Prior to sectioning, muscles were equilibrated at 20C for 1 hour. A

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30 microtome cryostat was then used to cut 10 m thick serial transverse sections, which were transferred to positively charge d glass slides Sections were allowed to dry for 1 hour at room temperature prior to freezing at 80 C until further processing or stained immediate ly using H&E to determine morphology. All stained sections were visualized using a Leica DM 5000B microscope (Leica Microsystems Bannockburn, IL ). Images of entire muscle cross sections were captured for 4 sections separated by at least 50 M per muscle Each histological analysis was pe rformed on at least 4 muscles per group. Hematoxylin & E osin (H&E) S taining H&E staining of muscle cross sections was performed as described previously (17) Briefly, slides were brought to room temperature prior to sequential submersions in the following solutions: 1 00% ethanol for 1 min, 70% ethanol for 1 min, dH 2 0 for 2 min Sections were then washed thoroughly in dH 2 0 seconds, dH 2 0 for 2 seconds, 70% ethanol for 1 minute, Eosin for 2 minutes, 95% ethanol with gentle shaking for 1 minute 100% ethanol for 30 seconds and Xylene for 2 3 minutes. Slides were allowed to dry for 30 minutes and then mounted with glass cover slips using Permount Von Kossa and T richrome S taining Slides containing fresh frozen muscle cross sections (10 m) were sent to the University of Florida Pathology Core Facility for t richrome staining to detect collagen (stains blue) or Von Kossa staining to detect calcium deposits ( stains black ).

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31 Immunohistochemistry For immunohistochemistry experiments, sections were permeabilized for 5 minutes in 0.1% Triton X 100, washed 2 X 5 minutes in PBS and blocked in Pierce Superblock for 15 minutes. Sections were then incubated in primary antibody diluted in blocking buffer at 4C overnight in a humid chamber. Primary an tibodies for the following proteins were used: embryonic myosin heavy chain ( 1:100, Developmental Studies Hybridoma Bank, Uni versity of Iowa, Iowa City, IA) and laminin (1:275, Sigma Aldrich) Sections were washed 3 X 5 minutes in PBS prior to incubation w ith the appropriate fluorescently conjugated secondary antibodies from Invitrogen in blocking solution for 1 hour at room temperature. CD68 AlexaFluor 488 conjugated primary antibody (1:175, AbD Serotec ), when used, was added during the secondary step. Sec tions were then washed 3 X 5 minutes in PBS and mounted with cover slips using VECTASHIELD fluorescence mounting medium with or without Dapi ( Vector Laboratories Inc ). BrdU I mmuno staining Muscle cross sections were rehydrated briefly in PBS prior to an tigen retrieval with citrate buffer (1.8mM citric acid and 8.2 mM sodium citrate, pH 6.0) as described previously (72) Endogenous peroxide activity was inhibited via incubating sections in 0.3% hydrogen peroxide for 5 minutes. Sections were washed in PBS and then incubated in primary antibody (anti BrdU, 1: 100 Roc he Dia gnostics, Indianapolis, IN, USA, and anti laminin, 1:275, Sigma Aldrich) overnight at 4C. The following day sections were washed extensively with PBS prior to incubation with secondary antibodies (Alexa Fluor 594 anti mouse IgG and Alexa Fluor 488 a nti rabbit IgG; Invitrogen) for 1 hour at room temperature. Sections were washed extensively, and then

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32 mounted with cover slips using VECTASHIELD fluorescence mounting medium containing D api (Vect or Laboratories ). Quantification of Muscle Necrosis Muscle R egeneration and Fiber CSA Q uantitative morphological analysis of TA and soleus muscle cross sections stained with H&E was performed on 4 serial cross sections ( separated by at least 50 M per muscle. R epresentative images from non overlapping fields were visualized and captured for each section using a Leica DM5000B microscope (Leica Microsystems Bannockburn, IL ). Fibers undergoing regeneration were identified as those containing centrally located nuclei Regenerating fiber cross sectional area (CSA) and the number of regenerating fibers in entire cross sections was calculated in muscles 16 days post cardiotoxin injection or following 10 days of muscle reloading, using Leica Application Suite software (version 3.5.0) This software was similarly used to ca lculate the average CSA of fibers in serial sections from muscles 28 and 42 days post cardiotoxin, except that the CSA of all fibers in entire cross sections were measured. Muscle fibers in early stage necrosis were identified on H&E stain as fibers with r educed eosinophilic staining and clear fiber borders with inflammatory cells in or surrounding the muscle fiber Muscle necrosis was quantified via counting and averaging the total number of necrotic muscle fibers per field in entire muscle cross section s in at least 4 serial sections separated by at least 50 m per muscle, n = 4 muscles per group. RNA Isolation, cDNA S ynthesis and qRT PCR Muscles to be used for RNA isolation were immediately removed, snap frozen in liquid nitrogen and stored at 80C Musc les were quickly minced in TRIzol reagent,

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33 1:10 (wt/volume), followed by thorough homogenization using a Polytron homogenizer and stored at 80C until further processing. Prior to RNA isolation, muscle homogenates were thawed to room temperature, vortexed thoroughly and RNA isolated using a chloroform directions as described previously (80) RNA concentrations (A 260 ) and quality (A 260 / 280 ) were assessed using a spectrophotometer Prior to qRT PCR, RNA (1ug) was reverse transcribed USA). Resulting cDNA was diluted 1/60 in dH 2 0 and used as a templ ate for real time PCR using universal primers purchased from Applied Biosystems. A 7300 real time PCR system (Appli ed Biosystems, Foster City, CA, USA) was used to detect PCR products and quantification was performed using a relative standard curve. RT 2 Profiler PCR Arrays RNA isolated as described above was reverse transcribed to cDNA using an RT 2 First Strand Kit fo g RNA as starting template (SA Biosciences ) RT 2 SYBR Green/ROX qPCR Master Mix and the Mouse Skeletal Muscle: Myogenesis & Myopathy PCR Array s (96 well Plates), both from SA Biosciences, were then used to am plify muscle specific gene transcripts from cDNA using a 7300 ABI PCR machine. Data was analyzed using the software provided by the manufacturer Because t he gene expression of the five house keeping genes on the PCR Array plates changed in response to card iotoxin treatment, we normalize d gene expression to histone deacetylase 5, which was not altered in respon se to cardiotoxin treatment, or between WT and Hsp70 / muscles during either condition

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34 Western Blot A nalyses Skeletal muscle protein extracts were prepared from gastrocnemius muscles from WT and Hsp70 / mice. Briefly, muscles were homogenized using a Dounce homogenizer in 1:10 (wt/vol) RIPA buffer ( 1% Triton X 100, 150 mM NaCl, 50 mM Tris HCl [pH 7.5], 0.1% SDS, 0.5% Na Deoxycholate ) plus protease i nhibitor cocktail and phosphatase inhibitors (Sigma Aldrich ). Homogenates were centrifuged at 5000g for 15 min at 4C and the supernatant collected. Protein content was assessed from supernatants using Bio Rad DC Protein Assay (Bio Rad Laboratories). Equal amounts of protein (30ug) were separated for 45 minutes on 4 15% gradient polyacrylamide gels containing 0.1% SDS using electrophoresis at 200V at room temperature. Separated proteins were electrotransferred to polyvinylidene fluoride membranes for 90 min utes at 100V in Tris Glycine buffer containing methanol at 4C. To prevent non specific binding, membranes were subsequently blocked in PBS tween (PBS T) containing 5% milk at room temperature for 1 hour. Membranes were incubated in primary ant ibody dilute d in blocking buffer (Hsp70, 1:500, Stressgen) overnight at 4C with gentle rocking. The next day membranes were washed extensively with PBS T followed by incubation in secondary antibody (LI COR Biosciences) for 1 hour at room temperature. Following exten sive washing in PBS T, membranes were rinsed in PBS and visualized using an infrared imaging system to detect and analyze labeled proteins (LI COR Biosciences). In vivo Muscle T ransfection and Rescue Experiments The Hsp70 EGFP expression plasmid was creat ed and used previously by our lab group (80) The EGFP expression plasmid was purchased from Clon etech In vivo plasmid DNA injection and electroporation has been described previously by our lab group (81) Briefly, 10 ug of Hsp70 E GFP or EGFP dissolved in 25ul PBS was injected

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35 along the longitudinal axis of the TA muscle using a 28 gauge insulin syringe. One minute following injection five electric pulses at 75 V/cm, duration of 20 ms, and interpulse interval of 200 ms were delivered to the muscle using an electric pulse generator (Electro Square porator ECM 830, BTX), by placing two paddle like electrodes on either side of the muscle Following experimental treatments and muscle harvest, EGFP and Hsp70 EGFP fluorescence was visualized in muscle cross sections using a Leica DM5000B microscope and a GFP filter. Statistical Analyses All data were analyzed using ANOVA followed by post hoc comparisons test when appropriate (GraphPad Software, San Diego, CA, USA). All

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36 CHAPTER 3 RESULTS AND DISCUSSI ON Genetic Deletion of Hsp70 Impairs S k eletal Muscle Fiber Growth The current study is the first to determine the effect of Hsp70 knockout on skeletal muscle plasticity Although we are specifically interested in the requirement of Hsp70 in regulating plasticity in post natal skeletal muscle, t o our knowledge, the phenotype of muscles which lack Hsp70 during embryonic growth and development has not been determined. Therefore, the use of lifelong Hsp70 / mice in the current study also allowed us to determine whether knockout of Hsp70 during deve lopment results in an overt muscle phenotype. Confirmation that Hsp70 / mice do not express Hsp70 was verified through qRT PCR ( Figure 3 1 A) on gastrocnemius muscles from WT and Hsp70 / mice using primers for Hspa1a and Hspa1b gene transcripts, which bo th code for Hsp70. Western blot analyses using an Hsp70 antibody was also performed to confirm the absence of Hsp70 protein ( Figure 3 1 B) using tubulin as a loading control. To determine whether Hsp70 / mice display normal muscle phenotype tibialis ant erior (TA) muscles from 4 week old WT and Hsp70 / mice were removed and processed for H&E analyses. As depicted in Figure 3 2 skeletal muscles from WT mice (A ) and Hsp70 / mice ( B ) were morphologically indistinguishable at 4 weeks of age on H&E stain M easurement of s keletal muscle fiber cross sectional area and to tal numbers of muscle fibers in entire TA cross sections from WT mice and Hsp70 / mice revealed no significant differences, ( data not shown ) These data indicate that Hsp70 is dispensable for normal embryonic skeletal muscle developmental and early post natal skeletal muscle growth, at least up until 4 weeks of age.

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37 Since post natal muscle growth and maturation in mice continues until approximately 12 weeks of age, we further compared fiber cro ss sectional area and fiber type composition in muscles from 12 week old WT and Hsp70 / mice. To do this, we immunostained muscle cross sections to identify Type I, Type IIa and Type IIb/x muscle fibers and measured CSA for each fiber type in entire cross sections n= at least 4 mice per group (Figure 3 3). This allowed us to determine fiber ty pe composition, total number of muscle fibers and average CSA for each fiber type. Although the soleus muscle is a predominately slow muscle, we chose to measure th ese variables in the soleus since it contains all three fiber types, which are distinguished based on the relative isoform of myosin heavy chain expressed in the muscle fiber. WT mice had a verage CSAs of 1457 m 2 for Type I fibers 1307 m 2 for Type IIa fibers and 2000 m 2 for Type IIb/x fibers, with an average fiber CSA of 1,588 m 2 across all muscle fibers Soleus muscles from Hsp70 / mice had average CSAs of 1157 m 2 for Type I fibers (21% smaller than WT p<0.05 ), 1063 m 2 for Type IIa fibers (19% smaller than WT p<0.05 ) and 1549 m 2 for Type IIb/x fibers (23% smaller than WT p<0.05 ), with an average fiber CSA of 1256 m 2 across all muscle fibers (17% smaller than WT p<0.05 ) ( Figure 3 3 B ). The average n umber of fibers for each fiber type in soleus muscles was not statistically different between WT and Hsp70 / mice (Figure 3 3 C). Therefore, fiber type composition was not altered in Hsp70 / mice Together these data indicate that while Hsp70 is dispens able for normal skeletal muscle development and post natal muscle growth up until 4 weeks of age, Hsp70 appears to play an important role in regulating skeletal muscle fiber size This discrepancy in muscle fiber size between WT and Hsp70 / mice may refle ct deficits in

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38 the later stage of skeletal muscle growth and maturation that occur s between 4 and 12 weeks of age in mice However, since we have previously demonstrated that Hsp70 overexpression repress es atrophy related signaling pathways in skeletal mu scle, the decreased skeletal muscle fiber CSA in Hsp70 / mice may be due to increased signaling through these atrophic pathways in the absence of Hsp70 even under normal physiological conditions Although the precise mechanism to explain this deficit in fiber size is not clear, these data demonstrate that Hsp70 is necessary for normal skeletal muscle fiber size under normal physiological conditions. Hsp70 / Mice have Impaired Muscle Regeneration In order to determine the requirement of Hsp70 for normal m uscle regeneration and recovery following injury, we injected TA muscles of 7 week old WT and Hsp70 / mice with 100 l of 10 M cardiotoxin which is known to result in widespread muscle fiber necrosis. This standardized method of muscle injury allows for the stud y of muscle fiber degeneration and subsequent muscle fiber regeneration. Muscles were harvested 1, 4, 16 28 or 42 days following cardiotoxin injury Analyses of muscle cross sections o ne day post injury via H&E demonstrate that TA muscles from WT mice showed signs of edema, inflammatory cell infiltration and myofiber necrosis as indicated by irregular eosinoph ilic staining of fibers with loss of normal fiber architecture (Figure 3 4 A). Four days post cardiotoxin necrotic fibers were largely cleared in WT muscles and replaced by mononuclear cells and central nucleated myoblasts, indicative of the early phase o f muscle regeneration (Figure 3 4 B ). In contrast, injured muscles from Hsp70 / mice one day post cardiotoxin showed less overt signs of fiber damage than WT, as visualized by fewer numbers of inflammatory cell infiltrates and preservation of fibers with normal

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39 eosinophilic staining and fiber borders However, despite this apparent decrease in muscle fiber damage one day post cardiotoxin Hsp70 / animals displayed marked abnormalities in morphology four days post cardiotoxin including persisting necrotic fibers with abnormal basophilic staining which appeared reminiscent of myofiber calcifications (8) H&E a nalysis of injured TA muscles from WT mice 16 days post c ardiotoxin revealed restoration of normal muscle fib er architecture and centrally nucleated regenerating muscle fibers (Figure 3 5 A) In contrast, TA muscles from Hsp70 / mice still presented with numerous necrotic muscle fibers and inflammatory lesions at this time point, and similarly displayed signs of myofiber calcifications seen in the Hsp70 / mice four days post cardiotoxin (Figure 3 5 B ) Importantly as shown in Figure 3 5 C the average cross sectional area of regenerati ng muscle fibers (those containing centralized nuclei) in Hsp70 / TA muscles ( 945 m 2 ) was significantly smaller than that of WT ( 1524 m 2 ), n=4 muscles/group, p<0.05 Furthermore, the average number of regenerating myofibers containing centralized nuclei in injured muscles was nearly 4 0% greater in Hsp70 / mice when compared to WT mic e a t this time point (Figure 3 5 D ). Presentation of this data as a frequency distribution ( Figure 3 5 E ) demonstrates that the increased numbers of regenerating fibers in muscles from Hsp70 / mice lie predominately in the CSA range of 250 750 m 2 (bin c enter = 500). This significant increase in small regenerating fibers in muscles from Hsp70 / animals at this time point could be the result of ongoing cycles of muscle degeneration/ regeneration However, this finding is also suggestive of decreased m yotu be fusion, since the muscles from Hsp70 / mice a lso had significantly lower numbers of fibers containing centralize d nuclei in the larger CSA range (>1500 m 2 ).

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40 Persistent Muscle Inflammation in Hsp70 / Mice f ollowing I njury Sustained and elevated muscle inflammation is well evidenced to interfere with muscle regeneration (1, 91) Based on the persisting presence o f mononuclear cells and necrotic muscle fibers seen in H&E stained sections from Hsp70 / muscles 16 days post cardiotoxin, we determined whether the Hsp70 / mice indeed showed increased markers of inflammation. To do this we stained for the macrophage ce ll surface marker, CD68 which is a transmembrane glycoprotein expressed on macrophages with a pro inflammatory (M1) phenotype S erial sections from WT and Hsp70 / muscles 16 d ays post cardiotoxin injury were incubated with antibodies for CD68 (green) and l aminin (red) which stains the basement membrane of muscle fibers As shown in Figure 3 6 A significant CD68 positive staining was seen across entire muscle cross sections from Hsp70 / but not WT mice. Magnification and separation of the overlayed ima ges to show CD68 and laminin staining separately in Figure 3 6 B, further demonstrates that the CD68 positive macrophages are present both in side and su rrounding muscle fibers Comparison of these CD68 stained areas to serial sections stained with H&E f urt her demonstrate that the abnormal basophilic sta ined fibers seen in the Hsp70 / mice are also positive for macrophages (corresponding fibers in CD68 and H&E stained sections are indicated with arrows). Further, fibers which appear infiltrated by numerous mononuclear cells (basophilic puncti) on H&E stain (outlined in white) also stain diffusely for CD68, demonstrating that these necrotic fibers are strongly infiltrated by pro inflammatory macrophages. The average number of CD68 positive cells (outside the basal lamina) and the average number of fibers which show positive staining for CD68 positive macrophages (inside the basal lamina) were quantified separately in entire muscle cross section s in WT and Hsp70 / mice 16 days post injury (Figure 3 6 C

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41 and D) Injured muscles from Hsp70 / mice showed an approximate 7 fold incr ease in both the number of CD68 positive macrophages surrounding muscle fibers and the number of muscle fibers infiltrated by CD68 positive macrophages, which further demonstrate s the m agnitude of inflammation still present 2 weeks post injury in muscles lacking Hsp70. These data suggest that Hsp70 is necessary for both normal muscle regeneration and resolution of the immune response following muscle injury. Calcification of Regeneratin g Muscle from Hsp70 / M ice following Injury Following damage to the muscle membrane as a result of muscle injury, intracellular calcium concentrations rise due to an influx of extracellular calcium (97) T he cardiotoxin induced model of muscle regeneration is believed t o induce muscle injury through disrupting ca lcium homeostasis causing depolarization and c ontracture of muscle fibers which results in mechanical injury to the muscle (43) Further, i ncreased levels of intracellular calcium also play important roles in initia ting muscle fiber proteoly tic pathways and dege neration (97) though calcium can also activate membrane re pair processes Importantly, this excess calcium is eventually cleared during the normal cycle of degeneration/ regeneration and calcium deposition does not occur. Based on the abnormal basophilic staining of muscle fibers in regenerating muscles from Hsp70 / mice which appeared similar to muscle fibers staining positive for calcium deposits in previous studies (Yi Ping Li), we determined whether these abnormal fibers were positive on Von Kossa stain, which stains calcium in black. Indeed, a s visualized through Von Kossa staining, which stains calcium deposits black WT muscles do not show calcium deposition 16 days post injury (Figure 3 7 A ) In contrast, muscles from Hsp70 / mice show pronounced calcium deposition in injured muscles 16 days post injury (Figure 7 A and B) When compared to an H&E stained

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42 serial section from the same area of the muscle it is clear that these calcium deposits are associated with inflammatory lesions and abnormal basophilic staining of fibers that in earlier experiments al so stained positive for CD68+ macrophages. l ncreased levels of calcium and c alcium dep osits are a hallmark of persiste nt muscle degeneration and fiber necrosis that is seen in dystrophic muscle phenotypes (97) Between the deficits in regenerating muscle fiber size, sustained presence of pro inflammatory macrophag es and development of c alcifications following injury, these data clearly demonstrate that mice lacking Hsp70 have significant impairments in m uscle regeneration and recovery following injury M uscles from Hsp70 / M ice S ho w A ltered G ene E xpression P rofil e 4 D ays P ost Cardiotoxin Injury The impairments in muscle regeneration seen in muscles of Hsp70 / mice 16 days post cardiotoxin may result from impairments in various aspects of the muscle degeneration/ regeneration process. Since marked abnormalities wer e noted in morphology as early as 4 days post car diotoxin inury we chose to compare the gene expression changes in response to cardiotoxin in muscles from WT and Hsp70 / mice 4 days post injury Since cardiotoxin injury of the muscle is localized to the area of injection, the uninjured, contralateral TA muscle was used as an internal control for each animal (n= 3 muscles/group ) The gene expression profile of regenerating skeletal muscle s following cardiotoxin injury has previously been demonstrated (100) Various genes are coordinately regulated during muscle regeneration including genes involved cell cycle control, inflammatory cytokines, matrix remodeling proteins and muscle specific transcription factors that ar e involved in muscle satellite cell activation and differentiation (100) We chose to utilize Myogenesis and Myopathy PCR arrays to

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43 compare the relative changes in gene expression in response to cardiotoxin in WT an d Hsp70 / animals. These arrays allow ed for the measurement of gene expression changes of 84 genes known to be differentially regulated in skeletal muscle during myopathy and muscle regener ation Since all housekeeping genes on the PCR arrays changed in r esponse to cardiotoxin, we chose to normalize gene expression to HDAC5 whose gene expression levels did not significantly differ between WT and Hsp70 / mice in uninjured TAs, or in response to cardiotoxin. Wh en comparing the magnitude of change in these genes between WT and Hsp70 / mice, numerous genes showed significantly higher magnitudes of activation in response to cardiotoxin in muscles from Hsp70 / mice (Figure 3 8 A C) Among these genes were pro inflammatory cytokines TNF (13 fold vs. 40 fold, p<0.05) and IL 6 (6 fold vs. 14 fold), growth factor IGF II (68 fold vs. 147 fold, p<0.05), the extracellular matrix remodeling protein, MMP 9 (21 fold vs 60 fold, p<0.05), and the calcium activated proteases, Calpain 2 (3 fold vs. 5 fold, p<0.05), Calpain 3 (0.9 fold vs. 2 fold, p<0.05) and Caspase 3 (19 fold vs. 32 fold, p<0.05). Similarly despite the deficits in regenerating fiber size seen in the Hsp70 / TA muscles 16 days post cardiotoxin, many of the typical markers of satellite cell activation, pr o liferation and differentiation in the Hsp70 / mice were either elevated or unaltered compared to WT The magnitudes of activation when comparing WT vs. Hsp70 / mice were as follows M yogenin (103 fold vs 259 fold, p <0.05 ), Myf5 (28 fold vs 40 fo ld ) Trop onin C (118 fold vs 401 fold, p<0.05) and Troponin T1 (58 fold vs 215 fold, p<0.05). Although Troponin C and Troponin T1 are typically thought as proteins expressed in adult slow muscle fibers, they are also highly expressed in myoblasts differentiating in to myotubes (65) Other myogenic markers including MyoD, Pax3 and

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44 Pax7 were not altered in Hsp70 / mice when compared to WT. To summarize, injured muscles from Hsp70 / mic e showed elevations in numerous proteins involved in muscle fiber degeneration and necrosis, inflammation as well as those which promot e regeneration. This quantitative gene data supports our histological findings in injured muscles from Hsp70 / mice whic h showed evidence for increas ed inflammation and pro inflammatory macrophages which are known to secrete both pro inflammatory cytokines a nd growth factors. Similarly, increased gene expression of the calcium regulated proteases involved in muscle protein breakdown in regenerating muscles from Hsp70 / mice is in line with o ur findings of calcium deposits that were strongly associated with degenerating myofibers and inflammatory lesions in these mice. In contrast, our finding that Hsp70 / mice have either increased or unaltered levels of proteins involved in activating the myogenic program was unexpected, as our h istological findings 16 days post injury demonstrate that Hsp70 / mice have deficits in muscle regeneration. Musc les from Hsp70 / Mice do not S how Deficits in Cellular Proliferation following Injury As many of the markers of satellite cell activation and proliferation were either unaltered or increased in Hsp70 / mice compared to WT, this suggests that Hsp70 / mice are not compromised in thei r ability to increase satellite cell proliferation following injury To further test this, WT and Hsp70 / mice were injected IP with BrdU dai ly following car diotoxin injury to label proliferating cells in regenerating muscles Six days post injury, the i njured TA and the contralateral, uninjured TA were harvested for histological analyses. Muscle cross sections from injured and uninjured TA muscles were co stained with antibodies for BrdU and laminin which stains the muscle fiber

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45 basement membrane (Figur e 3 9 ) Cross sections from injured TA muscles showing positive BrdU staining were also co unterstained with Dapi, to confirm the nuclear location of BrdU. Uninjured muscles from WT and Hsp70 mice showed relatively few BrdU positive nuclei. In contrast, i nj ured muscles from WT and Hsp70 / animals show ed abundant levels of BrdU nuclear staining in the center of muscle fibers, which is indicative of satellite cell p roliferation and differentiation (Figure 3 9 B) Although some BrdU positive nuclei may be thos e of inflammatory cells endothelial cells or other proliferating cells present in skeletal muscle, the consistent location of these BrdU positive nuclei in the center of regenerating myo fibers outlined by laminin strongly suggests that many of these nucle i indeed were derived from sate llite cell proliferation Therefore our findings using BrdU labeling of proliferating cells are in agreement with our gene expression data, which indicate that Hsp70 / mice do have deficits in satellite cell proliferation a nd differentiation, desp ite the deficits in regenerating fiber size in these mice two weeks post injury. B as ed on the significant elevation in the expression of the pro inflammatory cytokine, TNF in cardiotoxin injured muscles from Hsp70 / mice, and the persisting levels of pro inflammatory macrophages in these muscles 16 days post injury the deficits in muscle regeneration in Hsp70 / animals may be related to impairments in resolving the pro inflammatory immune response. Indeed, persisting levels of TNF and other pro inflammatory cytokines known to be released by pro inflammatory macrophages have inhibitory effects on muscle regeneration and recovery. Furthermore, since injured muscles from Hsp70 / mice also developed calcium deposits following injury, this indicates that these muscles may have ongoing calcium regulated

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46 proteolysis and fiber degeneration, which is supported by our histological findings 16 days post injury. Therefore, ongoin g muscle fiber degeneration in mice lacking Hsp70 may also interfe r e with successful regeneration. Muscles from Hsp70 / S how Impaired Muscle Recovery up to 6 weeks P ost I njury Persiste nt levels of inflammation and failed regeneration in skeletal muscle is known to result in replacement of muscle tissue with fibrotic tissue. Based on our findings that TA muscles from Hsp70 / m ice recovering from cardiotoxin inj ury show much higher levels of pro i nflammatory macrophages calcifications and smaller regenerat ing fibers we hypothesized that these mus cles would also show increased collagen deposition, which results in fibrosis. Trichrome staining of muscle cross sections 16 and 28 days post cardiotoxin injury which stains collagen blue, indeed demonstrates i ncr eased collagen deposition around muscle fibers in Hsp70 / muscles when compared to WT at both time points (Figure 3 10 A and B ). Quantitative a nalyses of fiber cross sectional area 28 days and 42 d ays post injury in WT and Hsp70 / mice provide further ev idence for the failed recovery of muscles following injury in the absence of Hsp70 (Figure 3 11). Indeed, at both of these time points, muscle fibers from Hsp70 / mice were still signif icantly smaller than those of WT (28 days post injury, WT = 2130 m 2 and Hsp70 / = 1251 m 2 p<0.05; 42 days post injury, WT = 1754 m 2 and Hsp70 / = 1461 m 2 p<0.05). Morphological analyses of muscles at the 42 day time point post injury through H&E staining further demonstrated that m uscles from Hsp70 / mice still d isplayed areas of inflammation, signs of fibrosis and calcium deposition, and clear deficits in fiber size

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47 At all time points measured post injury, regenerating myofibers in Hsp70 / mice did not re grow to a comparable size to regenerating myofibers in WT mice. These data therefore indicate that Hsp70 / mice have impairments in the processes which allow for normal muscle fiber regeneration and re growth following muscle injury. Muscle regeneration and growth following injury is a multistep process which involves satellite cell activation, proliferation, differentiation and fusion of myoblasts to regenerate multinucleated myofibers (7) Since Hsp70 / mice did not show deficits in the typical markers o f satellite cell activation, proliferation and differentiation following injury these data indicate that muscles from Hsp70 / mice may have deficits in the later phases of muscle regeneration that allow for myofiber growth. Importantly, fusion of differentiated myoblasts is an important event which both augments myofiber size and subsequently decreases fiber number. Since Hsp70 / mice also showed increased num bers of regene rating myofibers, in addition to decreased CSA of these regenerating fibers, we further suspect that Hsp70 / mice may have deficits in myotube fusion Although the detailed experiments needed to confirm this hypothesis are beyond the scope of the current study, this certainly warrants further investigation. Muscle Regrowth and R egeneration following R eloading induced I njury is Impaired in Hsp70 / M ice While our cardiotoxin s tudies clearly demonstrate that Hsp70 / mice have deficits in muscle regeneratio n and recovery following muscle injury, we sought to determine whether this would hold true in response to a more physiologically relevant form of muscle injury. One model of physiological muscle injury is that of muscle reloading injury (also referred to as modified muscle use) which occurs during the reambulation period following a prolonged period of muscle unloading or muscle disuse (17) For

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48 reasons largely unknown, following muscle disuse due to hind limb immobilization or hind limb suspension although the soleus, plantaris and gastrocnemius muscles all atro phy, upon reambulation only the soleus muscle demonstrates significant reloading damage (17) We therefore cast immobilized the hind limbs of WT and Hsp70 / mice for 10 days, removed the casts and allowed the mice to reambulate for either 0, 3 or 10 days prior to tissue harvest Muscle morphology was assessed at e ach time point through H&E staining of muscle cross sections (Figure 3 12 A). Immediately following cast removal (0 days reloading) soleus muscles from neither WT nor Hsp70 / mice show ed evidence of muscle damage. However, f ollowing 3 days of skeletal mus cle reloading, WT and Hsp70 / muscles showed evidence of edema, inflammatory cell infiltration and muscle fiber necrosis Early stage necrotic fibers, as identified on H&E stain as those fibers containing reduced eosinophilic staining with inflammatory ce lls infiltrating or surrounding the fiber, were minimally present in WT muscles (Figure 3 1 2 B ). N ecrotic fibers in WT muscles were either in late stage necrosis (heavily infiltrated by inflammatory cells and devoid of clear fiber borders) or cleared and r eplaced by mononuclear cells However, in muscles from Hsp70 / mice these early stage necrotic fibers were visually increased, and quantified to be ~7 fold higher than in WT muscles (Figure 3 12 B ), n = 4 mice per group, p<0.05. Although this may be inte rpreted such that muscles from Hsp70 / mice show increased muscle reloading damage, this did not appear to be the case. Indeed, despite the increased numbers of necrotic fibers in the 3 day reloaded Hsp70 / muscles, there were also more intact muscle fib ers remaining at this time point in the Hsp70 / mice when compared to WT. We hypothesize that muscles from Hsp70 / mice have a delayed and /or impaired muscle injury response,

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49 thereby resulting in delayed appearance and subsequen t clearance of necrotic mu scle fibers. Importantly, following 10 days of muscle reloading, similar to our findings using cardiotoxin injur y, muscles from WT mice had largely regained normal muscle fiber architecture and displayed many centrally nucleated fibers, while damaged areas from Hsp70 / mice were not recovered. Indeed, 10 day reloaded muscles from Hsp70 / mice showed abnormal muscle fiber architecture consisting of small rounded fibers hypercontracted or swollen muscle fibers (indicated by white arrow in the bottom panel of Figure 3 12 A ) and increased space between individual muscle fibers. Although the average number of fibers containing centralized nuclei in 10 day reloaded muscles were much lower in Hsp70 / mice when compared to WT (Figure 3 12 C ) quantification of f iber size in those fibers which did contain centralized nuclei revealed regenerating fibers in muscles from Hsp70 / mice were significantly smaller than those from WT muscles (Figure 3 12 D ) Representation of the CSA of regenerating fibers as a frequency distribution further demonstrates a significant left shift in the Hsp70 / mice, showing increased numbers of small, regenerating fibers, and decreased numbers of larger, regenerating fibers (Figure 3 12 E) Since we found in our cardiotoxin experiments t hat injured muscles from Hsp70 / mice showed elevations in both inflammatory and regenerative markers when compared to WT, we determined whether this would similarly be true in response to reloadi ng injury. To assess inflammation, cross sections from 3 da y reloaded muscles were stained with antibodies for the pro inflammatory macrophage marker CD68 (green fluorescence) and laminin (red fluorescence), which stains muscle fiber basement membranes Representative cross sections shown in Figure 3 13 demonstrat e s that WT

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50 and Hsp70 / muscles both showed significant levels of CD68 positive macrophages at this time point. C ross sections from 3 day reloaded muscles were also stained with embryonic myosin heavy chain ( e MHC) (red fluorescence), which is transcribed b y de novo formed myonuclei following satellite cell activation and differentiation, to assess the relative levels of regeneration in muscles at this t ime point (Figure 3 1 3 B ). Co staining with dapi, which stains nuclei blue, helped to visualize the e MHC p ositive myo blasts and myofibers. Of note, muscles from WT mice showed numerous e MHC positive myoblasts but few EMHC positive myofibers at this time point. In contrast, Hsp70 / mice showed numerous e MHC positive my ofibers. Analysis of gene expression in m uscles following 3 days of muscle reloading allowed for a more quantitative measurement of inflammatory and regenerative markers in WT and Hsp70 / mi ce, and is shown in Figure 3 1 4 Muscles from Hsp70 / mice demonstrated significant elevations in the mRN A levels of regenerative markers MyoD and myogenin, and inflammatory markers CD68 and TNF when compared to WT in 3 day reloaded muscles. Hsp70 / mice also showed increased mRNA levels of the prototypical NF B family member, p65, which is involved in classical NF B activation a nd pro inflammatory signaling. Interestingly, iNOS, which is oft en used as a marker of skeletal muscle damage due to its role in NO mediated damage of muscle fiber membranes, was significantly decreased in muscle s from Hsp70 / mice under control conditions, and its activation was similarly blunted in 3 day reloaded mu scles. Inhibition of nitric oxide production following muscle injury was recently shown to interfere with muscle regeneration and increase collagen deposition (15) Therefore, decreased iNOS and nitric oxide signali ng in Hsp70 / mice could play a role in both the decreased and/or delayed muscle injury

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51 response observed in Hsp70 / mice in the first few days following injury, as well as the impairments in muscle regeneration and recovery seen in these mice at later t ime points. Data collected thus far demonstrate that Hsp 70 / mice indeed have deficits in muscle regeneration and recovery following two distinct modes of muscle injury, cardiotoxin induced injury and mechanical injury due to modified muscle use In respo nse to both experimental models of muscle injury, muscles from Hsp70 / mice had elevations in inflammatory processes a nd defects in muscle recovery. Importantly, these deficits in muscle recovery included decreased size of regenerating myo fibers. However, muscles from Hsp 70 / mice did not show deficits in the normal regenerative markers during the early phase of muscle regeneration, including BrdU incorporation as a measure of proliferative capacity, MyoD and myogenin mRNA levels and eMHC expression. The refore, the deficits in regenerating fiber size in Hsp70 / mice may instead be related to deregulation of the inflammatory response which may subsequently impair fiber regeneration Terminal differentiation and growth of regenerating fibers in the later p hase of muscle regeneration in volves fusion of myoblasts to create multinucleated myotubes Importantly fusion of myoblasts can be repressed by pro inflammatory cytokines such as TNF and IL 6 (37, 38, 98) two cytokines which were elevated in injured muscles from Hsp70 / mice above WT. Further, pro inflammatory macrophages are a major source of these cytokines in injured muscle, and Hsp70 / mice showed elevations in these pro inflammatory macrophages up to 16 days post injury. Therefore it seems plausible that sustained inflammation in muscles of Hsp70 / mice may contribute to the defic its in regenerating fiber size through

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52 inhibiting myobla st fusion. Importantly TNF and IL 6 also have positive effects on muscle regeneration through their proproliferative action on satellite cells in the ear ly days following muscle injury (41, 98) Therefore, the increased expression of TNF and IL 6 in injured muscles 4 days post injury in Hsp70 / mice may also explain the ele vations in regenerative markers seen in these mice at this time point. Interestingly, iNOS mediated production of NO from muscle fibroblasts was recently shown to promo te myoblast fusion during post natal skeletal muscle growth and maturation (12) Since we saw significant reductions in iNOS mRNA in muscles from Hsp70 / mice during both control conditions and following muscle injury, it may be speculated that decreased NO signalin g (and therefore decreased myoblast fusion) may contribute to the increased numbers of myofibers with smaller CSA in regenerating muscles from these mice Restoration of Hsp70 in Skeletal Muscle Restores Regenerative Deficits in Hsp70 / M ice Since Hsp70 whole body knockout mice were used in the current study, the deficits in muscle regeneration and recovery may be due to the lack of Hsp70 in any cell type involved in muscle regeneration, including muscle fibers, satellite cells endothelial cells, fibrob lasts, inflammatory cells and other cells residing in skeletal muscle tissue. To determine whether restoration of Hsp70 levels in skeletal muscle fibers specifically can prevent the deficits in regeneration in the Hsp70 / mice we performed rescue experim ents via plasmid injection and electroporation of skeletal muscle with Hsp70 EGFP or EGFP expression plasmids. This method has been used extensively by numerous lab groups to induce transgene expression specifically in skeletal muscle fibers (70, 75, 80, 95) Hsp70 EGFP or EGFP plasmids were injected and

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53 electroporated into Hsp70 / TA muscles either 4 days prior to cardiotoxin injection or 4 d ays post cardiotoxin injection. As we observed abnormal gene expression an d morphology in Hsp70 / mice as ear ly as 4 days post cardiotoxin, this suggested to us that Hsp70 / mice may have significant impairments in the coordinated event s which occur during the first few days following muscle injury that allow for successful re generation. Therefore, electroporation of Hsp70 E GFP into the muscle prior to cardiotoxin injury aimed to test the importance of Hsp70 expressed in muscle fibers at the onset of muscle injury on the regulation of muscle re generation and recovery In contr ast, electroporation of Hsp70 E GFP 4 days following cardiotoxin injection allows for the transduction of newly regenerating myotubes and myofibers Therefore, the importance of Hsp70 in newly regenerating muscle fibers can be assessed from the fourth day f ollowing injury and beyond Comparisons on muscle recovery can then be made between Hsp70 / muscles transfected with Hsp70 GFP prior to cardiotoxin and following cardiotoxin injury to help delineate if and when muscle fiber derived Hsp70 contributes to su ccessful regeneration and recovery As shown in Figure 3 15 Hsp70 / muscles transfected with EGFP prior to cardiotoxin injury still demonstrated heavy infiltration of inflammatory cells signs of fibrosis and small regenerating fibers when visualized 16 days post injury In contrast, transfection of Hsp70 / muscles with Hsp70 EGFP prior to cardiotoxin injury largely restored the regenerative deficits seen in the Hsp70 / muscles The se Hsp70 EGFP transfected muscles from Hsp70 / mice were almost complet ely de void of inflammatory lesions and signs of fibrosis and contained visually larger regenerating fibers than Hsp70 / muscles transfected with EGFP.

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54 Analyses of TA muscles that were transfected with Hsp70 EGFP (or EGFP) 4 days post cardiotoxin, revealed only partial rescue to regenerating Hsp70 EGFP transfected muscle fibers (Figure 3 16) While Hsp70 EGFP positive fibers were visually larger than EGFP positive fibers rescued 4 days post injury, numerous inflammatory lesions were still present throughou t both the EGFP and Hsp70 EGFP transfected muscles. Therefore, the decrease in inflammatory cells seen in muscles transfected with Hsp70 EGFP prior to cardiotoxin injections was not visually apparent in muscles rescued with Hsp70 EGFP 4 days post cardiotox in. Together these data indicate that muscle derived Hsp70 is indeed necessary for normal muscle regeneration, and that Hsp70 play s an especially critical role during the first 4 days following cardiotoxin injury to promote a timely resolution of the infl ammatory response. The release of muscle fiber derived proteins to the extracellular space following membrane damage i n the first few days following muscle injury is believed to play an important role in activating and recruiting inflammatory cells to the site of muscle injury (91) As Hsp70 has been shown in numerous studies to participate in both the innate and adaptive immune responses (84) based on our findings it seems plausible that Hsp70 and/or an Hsp70 chaperoned peptide released from injured muscle may participate in the immune response that proceeds mu scle injury which has been speculated on previously (42) Interestingly, Prakken et al demonstrated several years ago now that injection of recombinant Hsp70 protein into rats was sufficient to induce anti inflammatory cytokines IL 10 and IL 4 in lymphatic cells, which is characteristic of a Th2 anti inflammatory immune response (69) IL 10 and IL 4 are known to activate M2 macrophages (25) which play an important role in resolving the pro inflammatory

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55 microenvironment in injured muscle through deactivating M1 pro inflammatory macrophages, and promoting muscle regeneration (91) Although we did not measure the levels of anti inflammatory macrophages or cytokines in the current study, it may be speculated that the increased and sustained pro inflammatory environment seen in muscles lacking Hsp70 are related to an imbalance in the M1/M2 macrophage phenotype Further research to delineate the mechanisms whereby muscle derived Hsp70 regulates the i mmune response is currently ongoing.

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56 Figure 3 1. Confirmation of Hsp70 mRNA and protein knockout in Hsp70 / mice. A) qRT PCR was performed on muscles from WT and Hsp70 / mice to confirm the absence of Hspa1a and Hspa1b gene transc ripts, which code for Hsp70. B) Western blot analyses using an antibody for Hsp70 was further performed to confirm the absence of Hsp70 protein, using anti tubulin as a loading control

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57 Figure 3 2. L ifelong Hsp70 knockout does not alter skeletal muscle development and morphology in 4 week old mice. A) Representative c ross sections of tibialis anterior (TA) muscles from 4 week old WT mice stained with H&E and visualized at high and low magnification to visualize individual muscle fibers and total muscle area B ) Representative cross sections of TA muscles from 4 week old Hsp70 / mice stained with H&E and visualized at high and low magnification to visualize individual muscle fibers and total muscle area

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58 Figure 3 3 Decreased fiber cross sectional area in soleus muscles of 12 week old Hsp70 / mice. A) Representative soleus muscle cross section s from 12 week old WT and Hsp70 / mice immunostained for Type I (blue), Type IIa (green) and Type IIb/x muscle fibers (black). B) A verage fiber cross sectional area (CSA) for Type I, IIa and IIb/x muscle fibers in soleus muscles from WT and Hsp70 / mice. C) Average number of Type I, IIa and IIbx muscle fibers and total number of muscle fibers in soleus muscles from WT and Hsp70 / mice. All data represent mean SE, n= at least 4 mice /group, *p<0.05.

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59 Figure 3 4 Abnormal morphology in Hsp70 / TA mus cles 4 days post cardiotoxin injury. A) Representative H&E stained cross sections from cardiotoxin injured TA muscles from WT and Hsp70 / mice 1 day post injury. B) Representative H&E stained cross sections from cardiotoxin injured TA musc les from WT and Hsp70 / mice 4 days post injury

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60 Figure 3 5 Muscles from Hsp70 / mice show defic its in regeneration 16 days post cardiotoxin injury A) Representative cross section from injured WT TA muscle demonstrating numerous regenerating musc le fibers and restoration of normal muscle architecture 16 days post injury. B) Representative cross section from injured Hsp70 / TA muscle showing impaired muscle recovery. C) Quantification of fiber CSA of fibers containing centralized nuclei (regenerat ing fibers) demonstrate that the CSA of regenerating fibers from Hsp70 / TA muscles are significantly smaller than WT 16 days post injury. D) Quantification of the average nu mber of regenerating fibers per section (x20 field view) in WT and Hsp70 / mice. E) Frequency distribution representing the percent of fibers at a given cross sectional area (+/ 250 m 2 ), with the exception of the bin center 0, which contains fibers from 0 250 m 2 All data represent mean SE, n= 4 muscles/group, *p<0.05.

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61 Figure 3 6 Muscles from Hsp70 / mice show increased numbers of CD68 positive macrophages 16 days post cardiotox in injury A) Representative cross section s from injured TA muscles from WT and Hsp70 / mice 16 days post injury stained with CD68 (green) to identify pro inflammatory macrophages and laminin (red) to outline muscle fiber basement membranes B) Magnifica tion and separation of the merged images (boxed areas) in (A) to better demonstrate the localization of CD68 positive macrophages to areas surrounding and inside of the basal lamina H&E staining of serial sections further confirm s that muscle fibers heav ily infiltrated by mononuclear cells on H&E stain are also positive for CD68 positive macrophages. C) Quantification of the number of CD68 positive macrophages outside of the basal lamina. D) Quantification of the number of muscle fibers showing CD68 stai ning (macrophages) inside the muscle fiber. All data represent mean SE, n=4 muscles/group, *p<0.05.

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62 Figure 3 7 Hsp70 / mice develop calcifications in muscles following cardiotoxin injury. A) Representative Von Kossa stained cross sections from cardio toxin injured WT and Hsp70 / TA muscles 16 days post injury demonstrate the presence of calcium deposits in injured Hsp70 / but not WT muscles. B) Magnification of the Von Kossa stained Hsp70 / TA c ross section (boxed area) is shown to better visualiz e the location of the deposits. An H&E stained serial section from the same area of the muscle demonstrates that the deposits identified in Von Kossa stain are associated with the dark basophilic stained areas and inflammatory lesions see on H&E stain. Corre sponding fibers in serial sections are indicated with asterisks

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63 Figure 3 8 Hsp70 / mice show altered gene expression 4 days post cardiotoxin injury Cardiotoxin induced changes in gene expression 4 days post cardiotoxin injury in WT and Hsp70 / mu scles are grouped by their magnitude of activation. A) Gene expression changes under 5 fold B) Gene expression changes between 5 and 50 fold C) Gene expression changes greater than 50 fold. Injured m uscles from Hsp70 / mice show enhanced activation of inflammatory cytokines proteases, growth factors, calcium regulated proteins and r egenerative markers compared to WT All data represent mean SE, n=3 muscles/group, *p<0.05.

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64 Figure 3 9 Measurement of BrdU positive proliferating cells in muscles from W T and Hsp70 / mice following cardiotoxin injury. A) Representative cross sections from uninjured TA muscles injected with BrdU daily for 6 days, and immunostained for BrdU (red) and laminin (green). B) Representative cross sections from injured TA muscles 6 days post injury from WT and Hsp70 / mice injected with BrdU daily beginning 6 hours prior to injury and immunostained for BrdU (red) and laminin (green). Sections were also counterstained with DAPI (blue), to demonstrate co labeling of BrdU with nucl ei (pink) in merged images

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65 Figure 3 10 Muscles from Hsp70 / mice show increased collagen deposition post cardiotoxin injury Representative cross sections from WT and Hsp70 / TA muscles 16 and 28 days post cardiotoxin injury stained with Trichrome to identify collagen deposition (blue). A) 16 days post cardiotoxin injury. B) 28 days post cardiotoxin injury.

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66 Figure 3 11 Regenerating muscles from Hsp70 / mice fai l to recover up to 6 weeks post cardiotoxin injury. A) Representative H&E stained cross sections of TA muscles from WT and Hsp70 / mice 42 days post c ardiotoxin injur y. B) The average CSA of muscle fibers in WT and Hsp70 / TA muscles was calculated 28 days and 42 days post cardiotoxin injury. Fibers from Hsp70 / TA muscles are signi ficantly smaller than WT at both time points post injury. All data represent mean SE, n=4 muscles/group, *p<0.05.

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67 Figure 3 1 2 Muscles from Hsp70 / mice show impaired regeneration and recovery following reloading injury. The hind limbs of WT and Hsp70 / mice were cast immobilized for 10 days, followed by cast removal and reambulation for either 0, 3 or 10 days. A) Muscle morphology was visualized via H&E staining. B) Quantification of t he average number of necrotic muscle fibers in 3 day reloaded musc les. C) Quantification of the average number of regenerating fibers containing centralized nuclei in 10 day reloaded muscles. D) Average CSA of regenerating fibers in 10 day reloaded muscles. E) Frequency distribution of regenerating fibers at a given CSA range in 10 day reloaded muscles. All data represent mean SE, n=4 mice/group, *p<0.05.

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68 Figure 3 1 3 Inflammation and regeneration in soleus muscles of WT and Hsp70 / mice following reloading injury. WT and Hsp70 / mice were exposed to 10 days of hind limb cast immobilization followed by cast removal and 3 days of muscle reloading. A) Inflammation was assessed in muscle cross sections through immunohistochemical staining for CD68+ macrophages (green) and laminin (red) which stains muscle fiber basement membranes B) Muscle f iber regeneration was assessed via immunohistochemical staining for embryonic myosin heavy chain (EMHC) (red), which is expressed by regenerating myofibers and differentiating myoblasts. Sections were counterstained with Dapi to demo nstrate that EMHC staining is localized to both small differentiating myoblasts and larger, regenerating myofibers.

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69 Figure 3 1 4 Altered g ene expression in muscles of Hsp70 / mice following reloading injury qRT PCR was performed in gastrocnemius musc les from WT and Hsp70 / mice exposed to 10 days of hind limb cast immobilization followed by cast removal and 3 days of muscle reloading. A) Relative mRNA levels of myogenic marker, MyoD. B) Relative mRNA levels of myogenic marker, Myogenin C) Relative m RNA levels of inflammatory marker, CD68 D ) Relative mRNA levels of iNOS. E) Relative mRNA levels of pro inflammatory cytokine TNF F ) Relative mRNA levels of the NF B transcription factor p65 All data represent mean SE, n=4 mice/group, *p<0.05.

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70 Figu re 3 1 5 Restoration of Hsp70 in muscle fibers 4 days prior to cardiotoxin inj ury rescues deficits in regeneration and recovery in Hsp70 / mice. TA muscles of Hsp70 / mice were transfected with either EGFP or Hsp70 EGFP and 4 days later injected with car diotoxin to induce muscle injury. A) Representative muscle cross sections 16 days post cardiotoxin injury showing EGFP and Hsp70 EGFP fluorescence under a GFP filter. B) S erial sections from injured muscles transfected with EGFP or Hsp70 EGFP prior to inju ry were stained with H&E to determine morphology 16 days post injury.

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71 Figure 3 1 6 Restoration of Hsp70 in regenerating muscle fibers of Hsp70 / mice 4 days following cardiotoxin in jury visually increases regenerating fiber size. Muscles from Hsp70 / mice were injected with cardiotoxin to induce muscle injury and 4 days following injury, were transfected with EGFP or Hsp70 EGFP plasmids. A) Representative EGFP and Hsp70 EGFP fluorescence in muscle cross sections 16 days post injury as visualized under a GFP filter. B) S erial sections from injured muscles injected with EGFP or Hsp70 EGFP were stained with H&E to visualize muscle morphology White asterisks denote corresponding muscle fibers in serial cross sections

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72 CHAPTER 4 CONCLUSIONS AND FUTU RE DI RECTIONS The current study used genetically modified mice which lack Hsp70 to test the hypothesis that Hsp70 is an indispensible protein necessary for normal skeletal muscle plasticity We found that skeletal muscles from 4 week old Hsp70 / mice were phen otypically similar to their WT counterparts, indicating that Hsp70 is not necessary for normal skeletal muscle development and early post natal muscle growth. However, by 12 weeks of age, when skeletal muscle growth and maturation comes to an end, muscle f ibers from Hsp70 / mice were at this point significantly smaller in CSA when compared to WT. Therefore, these data suggest that Hsp70 may be necessary for the later stages o f muscle growth and maturation. In addition we also found that Hsp70 / mice had significant deficits in muscle re growth and recovery following muscle injury that persisted up to 6 weeks post injury. These deficits in recovery in Hsp70 / mice were char acterized by smaller CSA of regenerating muscle fibers enhanced and sustained musc le inflammation and necrosis calcification of myo fibers, and collagen deposition, which are all characteristics of a dystrophic muscle phenotype. Successful m uscle regeneration and re growth of myofibers requires the activation and proliferation of satel lite cells and their commitment to myogenic differentiation (7) Based on both gene expression analyses of proteins inv olved in satellite cell proliferation and differen tiation and BrdU cellular proliferation assays post injury, Hsp70 / mice do not appear to have deficits in their ability to activate the myogenic program. However, successful growth of de novo myofibers n ot only requires satellite cell activation and differentiation, it also requires successful myoblast fusion to create the multi nucleated myofibers that span the length of the muscle. Therefore, the deficits

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73 in regenerating myofiber size seen in Hsp70 / m ice in the current study may be related to deficits in myoblast fusion. Although we did not quantitatively measure myoblast fusion in the current study, data collected in Hsp70 / mice during rescue experiments support this notion. Indeed, t he number of c entralized nuclei per myofiber (which measures fusion index) in Hsp70 E GFP positive myofibers appeared to be visually greater than that of E GFP positive muscle fibers from Hsp70 / TAs when visualized 16 days post injury. Since myoblast fusion supports myo fiber regrowth, and regenerating Hsp70 E GFP positive myofibers were also visually larger than E GFP positive myofibers at this time point, we hypothesize that Hsp70 supports myofiber growth in part, through promoting myoblast fusion. Although the detailed m echanisms to support this hypothesis were beyond the scope of the current study, this certainly warrants further study. Importantly, in addition to the deficits in regenerating muscle fiber size, Hsp70 / mice also showed significant elevations in pro infl ammatory markers persisted at numerous time points post injury While the specific mechanism behind the increased and sustained inflammatory signaling in regenerating muscles from Hsp70 / mice was not defined in the current study, rescue experiments in wh ich Hsp70 was restored in muscles prior to injury suggest that it may be related to impairments in the sequence of events which occur during the first 4 days following muscle injury. In this regard, rescue experiments in which Hsp70 E GFP was transfected in to skeletal muscle fibers 4 days prior to cardiotoxin injury prevented the inflammation and fiber necrosis seen in regenerating muscles of Hsp70 / mice 16 days post injury. In contrast transfection of Hsp70 E GFP into muscles 4 days post cardiotoxin injur y did NOT prevent the

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74 inflammation and f iber necrosis seen at this time point despite the positive affect it had on increasing myofiber size Therefore, we can conclude from these experiments that in addition to Hsp70 promoting growth of regenerating myo f ibers between days 4 and days 16 post injury, Hsp70 expressed in muscle fibers at the time of muscle injury also promotes resolution of the inflammatory response through a mechanism which occurs during the first 4 days following muscle injury. It is well e stablished that the pro inflammatory immune response that occurs in skeletal muscle during the first few days following muscle injury i s subsequently counteracted and resolved in part through a shift in the macrophage population towards an anti inflammator y phenotype These anti inflammatory or M2 macrophages secrete anti inflammatory, or Th2 cyto kines which promote muscle regeneration and repair (91, 92) Interestingly, evidence exists to support Hsp70 as an activat or of the Th2 immune response. Indeed, pre immunization or injection of recombinant Hsp70 into the foot pads of mice is sufficient to induce an anti inflammatory or Th2 response in immune cells (69) Therefore, it may be speculated that Hsp70 and/or an Hsp70 chareroned peptide derived from injured muscle fib ers may similarly promote an anti inflammatory response in regenerating muscle following injury to promote muscle recovery. Future research to delineate the mechanisms in which skeletal muscle derived Hsp70 regulates the immune response in regenerating mus cle following muscle injury is currently underway. In summary findings from th e current study demonstrate that Hsp70 is necessary for normal muscle regeneration and recovery. As experiments using muscle specific Hsp70 transgenic mice have previously demon strate d that Hsp70 overexpression can enhance muscle regeneration and functional recovery following injury, the results from

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75 this study provide further evidence to support the development and use of Hsp70 targeted therapeutics for a wide variety of skeleta l muscle disorders in which muscle regeneration is compromised. Importantly, our findings suggest that induction of Hsp70 in skeletal muscle both prior to muscle injury and during the growth phase of muscle regeneration may op timally promote successful mus cle regeneration and recovery.

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85 BIOGRAPHICAL SKETCH Sarah Marie Senf graduated cum laude from the University of Florida with a Bachelor of Science degree in microbiology and cell s cience in December 2006. She then immediately went on to pursue a Ph.D. in exercise physiology f rom t he University of Florida in January 2007 Sarah became a National Institute of Health T32 fellow in the Neuromu scular Plasticity Training Program at the University of Florida in 2008. Throughout her Ph.D. training, her research largely focused on understanding the molecular events which regulate skeletal muscle mass during atrophic conditions. Her early work demons trating the protective role of Hsp70 on muscle mass during atrophic conditions led to her dissertation project which focused on the requirement of Hsp70 during skeletal muscle regeneration and recovery following injury.