THE INFLUENCE OF SEX HORMONES ON CARDIAC AND SKELETAL MUSCLE FUNCTION IN THE MDX MOUSE MODEL OF DUCHENNE MUSCULAR DYSTROPHY

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1 THE INFLUENCE OF SEX HORMONES ON CARDIAC AND SKELETAL MUSCLE FUNCTION IN THE MDX MOUSE MODEL OF DUCHENNE MUSCULAR DYSTROPHY BY CONNIE BOOKLESS BSc (with Distinction) PGDipAppSci PhD Submitted for the completion of the degree of DOCTOR OF PHILOSOPHY At The Department of Biological and Physical Sciences Faculty of Sciences University of Southern Queensland 2006

2 ABSTRACT Duchenne Muscular Dystrophy (DMD) is a fatal recessive genetic human disease affecting one in 3500 live male births. DMD is progressive, there is no cure, and patients typically die of respiratory or cardiac failure in their second decade of life. Clinical disease symptoms are exacerbated at the onset of puberty and the physiological basis of this is unknown. The mdx mouse is the preferred experimental animal model of DMD, although aspects of the model remain poorly understood. This dissertation characterises physiological and histological features of the dystrophic mdx mouse in response to manipulations of hormonal status including testosterone treatment, surgical castration, and oestrogen treatment. Sex-specific differences in the mdx were also examined. Furthermore, physiological and histological features of the dystrophic mdx mouse model throughout the mdx lifespan were evaluated. Cardiac muscle contractility, left atrial response to exogenous calcium, and the contractile properties of both fast-twitch (EDL) and slow-twitch (SOL) skeletal muscles were examined in male mdx mice ranging from 14 to 330 days of age. Testosterone treatment produced a nonsignificant trend towards a dose-dependent decrease in both basal and maximal left atrial contractility in the mdx. Surgical castration produced no significant cardiac effects within mouse strains. The mdx castrates had a 45% lower maximum atrial force of contraction than control castrates (p<0.05). Conversely, oestrogen treatment significantly improved cardiac contractility in the mdx. An increase in basal left atrial contractility was evident at doses of 0.08 mg/kg/day (p<0.05) and 0.16 mg/kg/day (p<0.01) and in maximum left atrial contractility at a dose of 0.16 mg/kg/day (p<0.01). Gender studies showed cardiac forces in mdx were not different between males and females at any age tested and that both sexes in mdx had a dampened cardiac responsiveness to exogenous calcium. Skeletal muscle function studies showed that castration produced a 25% increase in mdx EDL specific force generation (p<0.01) and no increase in SOL forces. Oestrogen treatment produced a non-significant trend towards increased EDL forces and a 29% increase in SOL specific force at a dose of 0.16 mg/kg/day (p<0.05). Gender studies revealed no differences between male and female mdx in terms of skeletal muscle force production. Further to the hormonal investigations, lifespan characterisation studies revealed that the mdx mouse showed reduced basal and maximal left atrial contractility specifically at ages 14 and 90 days (p<0.05). Skeletal muscle studies showed that specific tetanic force production was significantly lower than controls at 19 (p<0.05), 21, 23, 27, and 330 days of age (p<0.01) for EDL muscles and at 19, 21, and 23 days of age in SOL muscles (p<0.01). These studies further improve our understanding of the mdx mouse as an experimental model of DMD and emphasises that the model is most appropriate a specific ages for specific muscles. These studies further illustrate that testosterone does not improve cardiac contractility in the mdx mouse but that oestrogen improves both cardiac and skeletal muscle function. Further research is warranted into the potential of oestrogen as a therapeutic agent in the treatment of both cardiac and skeletal muscle manifestations of DMD. i

3 CERTIFICATION OF DISSERTATION This thesis is my own work, except where otherwise acknowledged, and the work is original and has not been previously submitted for any other award at any other University or Institution of Tertiary Education. Signature of Candidate Date ENDORSEMENT Signature of Principal Supervisor Date ii

4 ACKNOWLEDGEMENTS This was a very personal and challenging journey and I wish to acknowledge and thank wholeheartedly the following people for their generosity in time and spirit that made the completion of this important work and subsequent dissertation possible. Firstly, to my supervisor Professor Andrew Hoey who has continued to inspire me from my earliest undergraduate studies with his passion for learning, his inspirational lecturing style, and ethical approach to research. To my husband Dr Gregory Bookless and our children, I love you and thank you. My friend and colleague Dr Christel van Erp, never change. To Dr Renee Cornford-Nairn and the members of the Muscle Research Laboratory USQ who always encouraged perserverance even in the toughest times. Special thanks go to Christine Bartlett (USQ Office of Research and High Degrees) who was just wonderful in her tireless encouragement, to Julie Murphy and Sandra Sharp whose professionalism and dedication in the USQ Animal House breeding facility ensured that my experimental animals were always cared for with the utmost concern for their welfare. In addition, the technical expertise and knowledge imparted by the following people greatly enhanced my skills and facilitated the completion of this project; Ilona Iberle (USQ Librarian), Paul Addison (UQ Histologist), Dr Peter Dunn (USQ Statistician), Oliver Kinder (USQ Technician), Maggie Fryer, Anne Woodroffe and Julie Pember (USQ IT Services), Debbie and Steven White (USQ Administrators), Chris de Byl, Phil Hallas, and Sammy Quatromani (USQ IT Services). I wish to make special mention of Mrs Debbie Robbins and Mrs Pat Furlong whose tireless work for the Duchenne Muscular Dystrophy Parent Project is an inspiration to us all. It is an honour to have known you and your courage and dedication moves mountains. Finally, and most importantly my love goes to every boy with DMD. You are the reason I strove for excellence and I hope that my work, in some small way, might just contribute to a better understanding of this devastating disease and may help to make your lives a little better. Motion is life.. Guillaume Benjamin Amand Duchenne de Boulogne iii

5 GLOSSARY OF ABBREVIATIONS AAV ACE AO AR BW C57 Ca 2+ [Ca 2+ ] i CHF CK CNF CO 2 CRC CSA CV cxmd DFZ DGC DHT DMD DNA ECG EDL enos EOM ER EtOH FOC FSH FXMD g GH adeno-associated virus angiotensin converting enzyme antisense oligonucleotide androgen receptor body weight C57BL/10ScSn mouse calcium intracellular calcium concentration congestive heart failure creatine kinase centronucleated fibre carbon dioxide concentration response curve cross-sectional area cardiovascular canine X-linked muscular dystrophy deflazacort dystrophin-associated glycoprotein complex dihydrotestosterone Duchenne muscular dystrophy deoxyribonucleic acid electrocardiogram extensor digitorum longus muscle endothelial nitric oxide synthase extraocular muscles oestrogen receptor ethanol force of contraction follicle stimulating hormone feline X-linked muscular dystrophy gram growth hormone iv

6 GI gastrointestinal GnRH gonadotrophin releasing hormone GRMD Golden Retriever muscular dystrophy H&E haematoxylin and eosin HFMD hypertrophic feline muscular dystrophy HR heart rate HW heart weight HW% heart weight as a percentage of body weight Hz Hertz IGF-1 insulin-like growth factor 1 inos inducible nitric oxide synthase IP intraperitoneal IQ intelligence quota K + kb kda KPSS LA Lf LH LIF Lo LV LV+S M MDSC mdx mg min ml mm mn msec potassium ion kilobase(s) kilo Dalton(s) Krebs physiological salt solution left atria/left atrium absolute muscle length leutinising hormone leukaemia inhibitory factor muscle length left ventricle/ventricular left ventricle and septum molar muscle-derived stem cell muscular dystrophy X-linked milligram minute(s) millilitre millimolar millinewton millisecond(s) µm micromolar v

7 µm micrometre n number of animals in experimental group Na + sodium ion NOS nitric oxide synthase nnos neuronal nitric oxide synthase NMJ neuromuscular junction O 2 PCR oxygen polymerase chain reaction % percent PK pyruvate kinase Po tetanic force Pt twitch force Po:Pt tetanus to twitch ratio RA right atria/right atrium REM rapid eye movement rpm revolutions per minute RV right ventricular free wall RyR ryanodine receptor SC subcutaneous SEM standard error of the mean SERCA sarcoplasmic reticulum calcium-atpase SHR spontaneously hypertensive rat SOL soleus muscle spo tetanic force normalised for cross-sectional area spt twitch force normalised for cross-sectional area SR sarcoplasmic reticulum TGF transforming growth factor TPSS Tyrode physiological salt solution U/L units of active enzyme per litre of serum USQ University of Southern Queensland VC vital capacity vs versus vi

8 CONTENTS ABSTRACT... I CERTIFICATION OF DISSERTATION... II ACKNOWLEDGEMENTS... III GLOSSARY OF ABBREVIATIONS... IV CONTENTS... IX LIST OF TABLES... XV LIST OF FIGURES... XVII 1 LITERATURE REVIEW History of Duchenne Muscular Dystrophy (DMD) Genetics and inheritance Detection, diagnosis and clinical disease features Genetic testing Serum creatine kinase (CK) and pyruvate kinase (PK) levels Muscle biopsy Disease onset Initial clinical signs Skeletal muscle involvement Biomechanics of movement in DMD Gowers manoeuvre Cardiac muscle involvement Skeletal system involvement Respiratory muscle involvement Smooth muscle involvement Intellectual and behavioural impairment Muscle and system sparing Female DMD carriers Serum CK and PK levels in female DMD carriers...19 vii

9 1.5.2 Female DMD carrier cardiac and skeletal muscle involvement Life expectancy and mortality Physical therapies Prolongation of ambulation Orthopaedic management of scoliosis Prevention of pulmonary mortality Prevention of cardiac mortality Dystrophin and the dystrophin-associated glycoprotein complex (DGC) Neuronal nitric oxide synthase and nitric oxide synthase Animal models of DMD The X-linked muscular dystrophy (mdx) mouse Alternative mouse models Feline disease models Canine disease models Zebrafish model The Caenhorhabiditis elegans worm Pathology and histology of DMD Calcium homeostasis Centronucleated fibres Therapeutic agents and strategies in DMD treatment and research Dampening the inflammatory response Corticosteroids Natural remedies Tumour necrosis factor-α Nitric oxide based therapeutics L-arginine Utrophin upregulation Improving protein synthesis Anabolic steroids Growth factors...49 viii

10 1.14 Decreasing proteolysis through reducing calpain activity β 2 -adrenergic agonists Increasing proliferation and regeneration of muscle cells Myostatin inhibition Leukaemia inhibitory factor Gene based therapies Aminoglycoside antibiotic therapy Antisense oligonucleotides Sex hormones and DMD Testosterone Oestrogens Aims of study METHODOLOGY Ethical considerations Experimental animals Treatment of animals - allometric scaling Individual trial methodologies Testosterone trial Sex (gender) trial Castration trial Oestrogen trial Influence of age trial Euthanasia Dissection of tissues Organ bath (functional) experiments Skeletal muscle experiments Cardiac muscle experiments Histology protocol Histological analysis Data analysis...76 ix

11 3 TESTOSTERONE TRIAL Influence of testosterone on body weight Influence of testosterone on testes weight Influence of testosterone on cardiac characteristics Cardiac morphometry Cardiac function Influence of testosterone on skeletal muscle morphometry Discussion SEX (GENDER) TRIAL Influence of sex on body weight and heart weight Influence of sex on cardiac characteristics Cardiac morphometry Cardiac function Influence of sex on EDL characteristics EDL morphometry EDL function EDL histology Influence of sex on SOL characteristics SOL morphometry SOL function SOL histology Discussion CASTRATION TRIAL Influence of castration on body weight Influence of castration on cardiac characteristics Cardiac morphometry Cardiac function Influence of castration on skeletal muscle morphometry Influence of castration on EDL characteristics x

12 5.4.1 EDL function EDL histology Influence of castration on SOL characteristics SOL function SOL histology Discussion OESTROGEN TRIAL Influence of oestrogen on body weight Influence on oestrogen on testes weight Influence of oestrogen on cardiac characteristics Cardiac morphometry Cardiac function Influence of oestrogen on skeletal muscle morphometry Influence of oestrogen on EDL characteristics EDL function EDL histology Influence of oestrogen on SOL characteristics SOL function SOL histology Discussion INFLUENCE OF AGE TRIAL Influence of age on body weight Influence of age on testes weights Influence of age on cardiac characteristics Cardiac morphometry Cardiac function Influence of age on skeletal muscle morphometry Influence of age on EDL characteristics EDL function xi

13 7.5.2 EDL histology Influence of age on SOL characteristics SOL function SOL histology Discussion CONCLUSIONS AND FUTURE DIRECTIONS REFERENCES APPENDIX I Krebs physiological salt solution (KPSS) recipe Tyrode s physiological salt solution (TPSS) recipe Haematoxylin stain (Mayer s Haematoxylin) recipe Eosin stain recipe Scott s solution recipe xii

14 LIST OF TABLES Table 1-1 Landmarks in the history of DMD... 2 Table 1-2 Reported manifestations in female DMD carriers...22 Table 1-3 Currently understood roles of the DGC...28 Table 1-4 Disease features of the GRMD canine model of DMD...37 Table 1-5 Therapeutic benefits and theoretical mechanism(s) of action of... corticosteroids in the treatment of DMD Table 1-6 Therapeutics agents in DMD treatment and research...53 Table 1-7 Known physiological effects of oestrogen(s) in non-dystrophic animals and humans...65 Table 2-1 Definitions and mathematical formulae used in skeletal muscle results calculations...74 Table 3-1 Weight of SOL and EDL muscles in male C57 and mdx mice at varying testosterone doses Table 4-1 EDL muscle function in 90 day old mice Table 4-2 EDL muscle function in 180 day old mice...98 Table 4-3 EDL muscle function in 330 day old mice...99 Table 4-4 SOL muscle function in 90 day old male and female mice Table 4-5 SOL muscle function in 180 day old male and female mice Table 4-6 SOL muscle function in 330 day old male and female mice Table 5-1 Body and cardiac weights in entire and castrated mice Table 5-2 Cardiac function of entire and castrated mice Table 5-3 Skeletal muscle morphometry of entire and castrated mice Table 5-4 EDL skeletal muscle function of entire and castrated mice Table 5-5 SOL muscle function in entire and castrated mice Table 6-1 Body and cardiac weights at varying oestrogen doses Table 6-2 EDL and SOL muscle morphometry at varying oestrogen doses Table 6-3 EDL muscle function at varying oestrogen doses Table 6-4 SOL muscle function at varying oestrogen doses Table 7-1 Body and cardiac weights of 14 day old male mice Table 7-2 Body and cardiac weights of 19 day old male mice Table 7-3 Body and cardiac weights of 21 day old male mice Table 7-4 Body and cardiac weights of 23 day old male mice Table 7-5 Body and cardiac weights of 27 day old male mice xiii

15 Table 7-6 Body and cardiac weights of 34 day old male mice Table 7-7 Body and cardiac weights of 40 day old male mice Table 7-8 Body and cardiac weights of 90 day old male mice Table 7-9 Body and cardiac weights of 180 day old male mice Table 7-10 Body and cardiac weights of 330 day old male mice Table 7-11 Skeletal muscle morphometry of 14 day old male mice Table 7-12 Skeletal muscle morphometry of 19 day old male mice Table 7-13 Skeletal muscle morphometry of 21 day old male mice Table 7-14 Skeletal muscle morphometry of 23 day old male mice Table 7-15 Skeletal muscle morphometry of 27 day old male mice Table 7-16 Skeletal muscle morphometry of 34 day old male mice Table 7-17 Skeletal muscle morphometry of 40 day old male mice Table 7-18 Skeletal muscle morphometry of 90 day old male mice Table 7-19 Skeletal muscle morphometry of 180 day old male mice Table 7-20 Skeletal muscle morphometry of 330 day old male mice xiv

16 LIST OF FIGURES Figure 1-1 Guillaume Benjamin Amand Duchenne de Boulogne and the first observed case of DMD... 2 Figure 1-2 Pathways of X-linked recessive inheritance in DMD... 4 Figure 1-3 Schematic diagram of the dystrophin gene Figure 1-4 Pseudohypertrophy of the calf muscles in DMD... 9 Figure 1-5 Classical Gowers manoeuvre (sign)...11 Figure 1-6 ECG pattern in a nine year old boy with DMD...12 Figure 1-7 Progressive body posture changes in DMD...13 Figure 1-8 Severe scoliosis of the spine in a boy with DMD...14 Figure 1-9 Dystrophin and the dystrophin-associated glycoprotein complex (DGC)...28 Figure 1-10 Vicious cycle of muscle degeneration in DMD...30 Figure 1-11 C57BL/10ScSn (C57) and X-linked muscular dystrophy (mdx) mice...32 Figure 1-12 Human skeletal muscle biopsies of unaffected and DMD affected individuals Figure 1-13 H&E stained sections of DMD affected human skeletal muscles Figure 1-14 H&E stained SOL muscles in a C57 and an mdx mouse Figure 1-15 Vector based therapeutic strategies in DMD...58 Figure 1-16 Oestrogen activation of enos Figure 3-1 Body weights of C57 and mdx mice at varying testosterone doses Figure 3-2 Testes weights of C57 and mdx mice at varying testosterone doses Figure 3-3 Left atrial weights of C57 and mdx mice at varying testosterone doses Figure 3-4 Basal force of contraction of left atria at varying testosterone doses Figure 3-5 Concentration response curves to calcium at varying testosterone doses Figure 3-6 Maximum force of contraction of the LA at varying testosterone doses Figure 4-1 Influence of sex on body weight and heart weight between strains and sexes Figure 4-2 Effects of age on cardiac tissue weights between strains and sexes Figure 4-3 Basal FOC of LA (normalised for LA weight) in mdx and C57 males and females across ages Figure 4-4 Concentration response curves to calcium comparing male and female 90 day old mice Figure 4-5 Concentration response curves to calcium comparing male and female 180 day old mice Figure 4-6 Concentration response curves to calcium comparing male and female mice 330 day old Figure 4-7 Maximum FOC of LA (normalised for LA weight) in male and female mice across ages xv

17 Figure 4-8 EDL skeletal muscle morphometry comparing mouse strains and sexes Figure 4-9 Percentage of CNFs and maximum tetanic forces in EDL muscles at varying ages and between sexes Figure 4-10 EDL cell diameters and maximum tetanic forces at varying ages and between sexes Figure 4-11 SOL skeletal muscle morphometry comparing mouse strains and sexes Figure 4-12 Percentage of CNFs and spo in SOL muscles at varying ages and sexes. 105 Figure 4-13 Cell diameters and spo in SOL muscles at varying ages and between sexes Figure 5-1 Influence of castration on body weight in C57 and mdx mice Figure 5-2 Concentration response curves to calcium in entire and castrated mice Figure 5-3 EDL percentage CNFs and spo for entire and castrated mice Figure 5-4 EDL cell diameter and spo between strains and for entire and castrated mice Figure 5-5 SOL percentage CNFs and spo between strains and for entire and castrated mice Figure 5-6 SOL cell diameters and spo between strains and for entire and castrated mice Figure 6-1 Body weights at varying oestrogen doses Figure 6-2 Testes weights at varying oestrogen doses Figure 6-3 Basal FOC of the LA (normalised for LA weight) at varying oestrogen doses Figure 6-4 Ca 2+ CRCs at varying doses of oestrogen Figure 6-5 Maximum FOC of the LA (normalised for LA weight) at varying oestrogen doses Figure 6-6 EDL percentage of CNFs at varying oestrogen doses Figure 6-7 EDL cell diameter at varying oestrogen doses Figure 6-8 SOL percentage of CNFs at varying oestrogen doses Figure 6-9 SOL cell diameter at varying oestrogen doses Figure 6-10 Pathways of testosterone action and the conversion to oestradiol Figure 7-1 Body weight comparison between C57 and mdx mice at varying ages Figure 7-2 Influence of age on testes weights across varying ages Figure 7-3 Basal FOC of the LA across ages Figure 7-4 Concentration response curves to calcium across varying ages Figure 7-5 Maximum FOC of the LA across ages Figure 7-6 SOL weight between strains across varying ages Figure 7-7 SOL CSA between strains across varying ages xvi

18 Figure 7-8 EDL weight between strains across varying ages Figure 7-9 EDL CSAs between strains across varying ages Figure 7-10 EDL spt across varying ages Figure 7-11 EDL spo across varying ages Figure 7-12 EDL percentage of CNFs and spo across varying ages in male C57 and mdx mice Figure 7-13 EDL cell diameters across varying ages Figure 7-14 SOL spt across varying ages Figure 7-15 SOL spo across varying ages Figure 7-16 SOL percentage of CNFs and spo across varying ages Figure 7-17 SOL cell diameters across varying ages Figure 7-18 Periods of peak skeletal muscle necrosis in the mdx mouse xvii