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Evaluation of muscle function in Six5 knock-out and (CTG) repeat overexpresser mice
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Evaluation of muscle function in Six5 knock-out and (CTG) repeat overexpresser mice
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EVALUATION OF MUSCLE FUNCTION IN SIX5 KNOCK-OUT AND (CTG) REPEAT OVEREXPRESSER MICE by Jyoti Nautiyal A Thesis Presented To the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (BIOCHEMISTRY) December 2004 Copyright 2004 Jyoti Nautiyal Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I express my profound gratitude to my advisor Dr. Sita Reddy, Department of Biochemistry and Molecular Biology, University of Southern California, for her invaluable assistance, precious guidance and constant encouragement throughout the work. The steady motivations and constructive criticism offered by her has not only helped me to complete my work in the present form, but also encouraged me to move on to Ph.D program in Detroit. It would have been my pleasure to continue my work towards a doctoral thesis in her lab, but for my married life, I have to move on. I am thankful to Dr. Reddy’s encouragement and all the support to continue my studies after marriage. This would always mean a lot to me as I move forward in life to accomplish new goals. I thank Dr. Personius for not only providing the advanced instrumental set-up but also guiding me through the compilation of this work. I would like to extend special thanks to other members of my committee for bearing with me in listening to my work and making it happen in time. Thank you Dr. Maxson and Dr. Tokes for taking out time from your busy routines to schedule this defense as per my needs. Thank you Dr. Tokes for all the help, talks, advice and suggestions to make things move smoothly. i i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thanks to all, I met at IGM and made a family for myself. I owe special gratitude to my seniors Dr. Sarkar, Dr. Paul, Dr. Dansithong for always being available and providing right directions to my experiments. Thank you Sherry Morcos and Agnes Banfalvi for your friendship and eagerness to help a foreigner settle down into the American life. Coming all the way from India, fighting homesickness during first two semesters was made so easy with your company and caring attitude. Thanks so much Agnes for your critical comments regarding everything we discussed. Last but not the least, I would extend my thanks to my bhayya and bhabhi and my hubby. Words would not be enough to express my gratitude to you all, for being with me through thick and thin. Your presence around me all the times meant so much to me. Praveena & Raveendra and Bibha & Sathees thanks for proving “friends in need are friends indeed”. Thanks Bibha and Sathees for being more than friends and providing me a family environment through out my stay in Los Angeles. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Acknowledgements ii List of Tables v List of Figures vi Abstract vii Chapter 1 Introduction 1.1 Myotonic Dystrophy 1 1.2 DM1 Locus, Transcripts and Proteins 8 1.3 Current approaches to dissect the DM1 molecular Pathogenesis 12 1.4 DM2- A Parallel universe 20 1.5 Hypothesis 21 Chapter 2 Materials and methods 2.1 Six5 knock out mice 24 2.2 (CTG) repeat overexpressor mice 26 2.3 Genotyping the mice lines by PCR amplification 29 2.4 Muscle electrophysiology 37 2.5 Muscle histology 41 Chapter 3 Results 3.1 Genotyping and Experimental set-up 44 3.2 Data analysis 51 Chapter 4 Discussion and Conclusion 41 Biological rationale 71 4.1 Conclusion 77 4.2 Future directions 78 References 81 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table (1.1) Systemic involvement in Myotonic Dystrophy 2 Table (1.2) Myotonic Dystrophy: Molecules and Signs 19 Table (1.3) DM1 vs. DM2: Comparative Features 22 Table (2.1) Strategy for genotyping Six5 knock out mice by PCR amplification 33 Table (2.2) Six5 amplification: PCR reagents, volume and concentration 35 Table (2.3) Kreb’s solution: Chemical Composition 39 Table (3.1) Six5 cDNA sequence 45 Table (3.2) Neomycin sequence 46 Table (3.3) Lac-Z sequence 48 Table (3.4) Contractile abnormalities are not present in Six5 deficient mice 63 Table (3.5) Contractile abnormalities are present in (CTG)7o o / (CTG)goo transgenic mice 69 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 1.1 1.2 1.3 2.1 2.2 2.3 3.1 3.2 3.3 3.4 3.5 3.5 3.7 3.8 3.9 Schematic representation of DM1 locus 4 Molecular basis of DM1 pathology 6 Schematic representation of genetic anticipation across three generations 7 A schematic diagram to show the Six5 knock out construct 25 Strategy for screening Six5 knock out mice 27 Ectopic expression of (CTG) repeats in skeletal muscle 28 Six5 PCR amplification 50 Neomycin PCR amplification 50 Striated appearance of a muscle 54 Sarcomere: The functional unit of a skeletal muscle 54 Muscle contraction: a response to single stimulus 56 Muscle Twitch: parameters 56 Muscle contractions: response to increasing frequency of stimulation 58 Muscle Tetany: parameters 58 H&E sections of soleus muscle 65 3.10) EMG: Demonstration of myotonia in (CTG)7 o o transgenic mice 67 VI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT DM1, muscular dystrophy 1 is characterized by hyperexcitability of skeletal muscle (myotonia) and muscle degeneration (myopathy). The cause for DM1 is found to be a (CTG) triplet repeat expansion in the 3’ UTR of DMPK gene. The present study investigated if the reduced levels of Six5 and (CTG) repeat over expression also lead to muscle weakness. Six5 knock-out mice did not show any myotonia or muscle weakness. (C T G )7oo/(CTG)g0o transgenic mice showed statistically significant decrease in force production and showed significant myotonia. The significant differences in the muscle function in the (C T G )7oo/(C TG )g0o repeat mice show that (C T G ) repeat expansion is central to the muscle abnormalities as observed among the DM1. The (CTG)goo mice show a milder muscle phenotype compared to the (C T G )7oo overexpresser mice because the of the low levels of the mRNA expression of the (CTG)goo transgene as compared to the (CTG)7 q o transgene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1 INTRODUCTION 1.1 Myotonic Dystrophy 1.1.1 The Disease Myotonic Dystrophyl (DM1), also known as dystrophia myotonica or Steinert disease, was first described about one hundred years ago (Batten and Gibb, 1909; Steinert, 1909: Harper 1995). DM1 is the most common form of muscular dystrophy among adults, with an estimated incidence of 1:8000 (Harper, 2001). It is a muscle disease that is striking in its variability among patients and its seemingly unconnected symptoms. It is characterized by hyperexcitability of skeletal muscle (myotonia) and muscle degeneration (myopathy). Although DM1 is primarily a progressive neuromuscular disorder, DM1 patients frequently exhibit additional characteristic non-muscle symptoms, including ocular cataracts, neuropsychiatry impairments, endocrine abnormalities, cardiac conduction defects, testicular atrophy and premature balding. Congenital DM constitutes a very severe disorder, characterized by high neonatal mortality and symptoms like hypotonia, mental retardation and respiratory distress. Table (1.1) gives a brief summary of the different systems involved in myotonic dystrophy. 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (1.1) Systemic involvement in myotonic dystrophy Organ or system Clinical Muscle Myotonia, weakness and dystrophy EMG: decreased resting membrane potential; repetitive depolarization. Pathology: sarcoplasmic masses, ringed fibers, internal nuclei, frequent; nuclei often in chains; large variation in fiber size Cardiac Bradycardia common; complete heart block frequent; prolonged PR interval First degree heart block, bradycardia on ECG; abnormal vectorcardiogram;SA node, left and right bundle branch dysfunction and increased HisPurkinje conduction (His bundle studies) Lens Posterior subcapsular, scintillating cataracts Dust like cataracts may be visible only on slit lamp examination Eye Decreased vision (independent of cataracts and diabetic retinopathy) Pigmentary disorders of macula, keratosis sicca; decreased intraocular pressure CNS Mental retardation; hypersomnolence Possible neuronal heterotopias. Suspicious, reticent personality characteristics Endocrine Abnormal carbohydrate metabolism; testicular (and ovarian) atrophy Abnormal glucose tolerance with elevated insulin levels. Gonadal fibrosis (pathology), decreased 17- keto steroids (occasional). Decreased metabolic rate, normal thyroid hormone levels Integument Frontal balding Calicifying epitheliomas Gastrointestinal Dysphagia and intestinal pain Disordered esophageal and gastric peristalsis; dilation of bowel Skeletal Cranial and facial abnormalities; malocclusion of dentition Cranial bony abnormalities, hyperostosis of skull, small sella turcica, large sinuses, micrognathia Respiratory Hyperventilation; Post anesthesia respiratory failure Diaphragmatic and intercostals muscle weakness Blood None Abnormal erythrocyte scanning electron microscopy. Decreased phosphrylation of specific erythrocyte membrane glycoprotein. Decreased ouabain-responsive sodium efflux. Increased fluidity of membrane Reference: Harper, P. S. in The Molecular Basis of Inherited Disease (1995). 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.2 Genetic Mapping of DM1 Inheritance of myotonic dystrophy (DM1, MIM 160900) is autosomal dominant, that is a mutation in one copy of the affected gene causes the disease. The gene affected in DM1 has been mapped to chromosome 19q13.3, and encodes for the myotonin protein kinase (DMPK). Nearly all affected DM1 individuals possess a (CTG) triplet repeat expansion in a region of the DMPK gene that corresponds to the 3’ UTR of the mRNA. Due to its position in a non-coding region, the (CUG) tract in the DMPK mRNA is not translated. Figure (1.1) shows the DM1 locus and the position of (CTG) repeat tract in the 3’ UTR of DMPK. 1.1.3 (CTG) triplet repeat and DM1 pathology The number of (CTG) repeats is variable ranging from 5-37 triplet repeats in normal cells to >700 repeats in the severe forms of the disease. Global disease severity and age of onset correlate reasonably well with repeat length in the blood: alleles in the 35-49 repeat range are considered premutation alleles, mild or very late onset form of the disease is seen in individuals with (CTG)5 o-i5o repeats, “classic” disease manifestation is associated with (CTG)ioo-5oo and in congenital DM 1 patients, the repeats contain several thousands of (CTG) triplet repeats (Groenen and Wieringa, 1998; Harper, 2001). Although there is an overlap in range of repeats between different forms of D M 1 , there is a rough correlation between the 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (CTG)n DMWD DMPK SIX5 WDmpeat protein serina/thmonim protein kinase homeoclomain transcription factor Schematic representation of the DM1 locus: The DM1 locus is strongly conserved between mouse and man and contains three genes, DMWD, DMPK and SIX5, located closely together in a gene-dense region. Transcription initiation sites are indicated with arrows; exons with boxes, and introns and intergenic sequences with a straight line; alternative splice modes with connecting lines between exons, and polyadenylation sites with asterisks (two for DMWD). Figure (1.1) Schematic representation of the DM1 locus. Reference: Wansink et al., Cytogenet. Genome Res., 2003 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. repeat number, the severity of phenotype and the reduction in the age of onset, as shown in Figure (1.2). Patients with moderate or long expansions demonstrate age- dependent, mosaicism in the repeat length, which is presumably mediated via multiple small length change events (Thornton et al., 1994; Monckton et al., 1995; Wang et al., 1995; Martorell et al., 1997). Besides being highly unstable in the germ line, the repeat length varies considerably within and between somatic tissues. For example, repeats in the myocytes (skeletal muscle and heart) are greater than those found in lymphocytes. Though a clear picture is not available currently to explain this mosaicism, the understanding of this phenomenon may provide a correlation between distribution of the average repeat length and the disease spectrum and severity. There is also an evidence of genetic anticipation (the progressive earlier onset of the disease and worsening of the symptoms in subsequent generations) and a tendency for maternal transmission of a severe congenital form of the disease. Above a critical threshold, the (CTG) repeat has a greater than 95% chance to expand from one generation to the next causing anticipation (Groenen and Wieringa, 1998; Harper, 2001). Figure (1.3) demonstrates the phenomenon of genetic anticipation and the severity of disease phenotype across three generations. 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The molecular basis of anticipation in DM is the expansion of a (CTG)n repeat on ch!9 Congenital DM (CTS)n = 500- >1000 Adult onset DM (CTS)n = 100-500 DM1 severity increases with progressive expansion of the CJ& tract Normal: (CT£)n=5-35 K 19ql3.3 Figure (1.2) Schematic diagram to show the severity of phenotype as a consequence of expansion of (CTG) repeat. 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Genetics Autosomal dominant disorder Genetic Anticipation Number* Severity of s ym pt om ! Increase Within a pedigree □ Mild-DM fa A Adult-onset DM ' • » - Congenital DM Figure (1.3) Schematic representation of Genetic anticipation of the DM1 across three generations. (Modified from Myology: by Andrew G. Engel, Clara Franzini-Armstrong, McGraw-Hill, Medical Pub. Division, c2004.) 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2 DM1 Locus, Transcripts and Proteins The (CTG) repeat is located within the last 3’ untranslated exon of the human myotonic dystrophy protein kinase (DMPK) gene, and is found just 5’ of the SIX5, which encodes for a homeodomain protein (Fig. 1.1). Immediately, upstream of the DMPK gene lies the DMWD gene. It has been shown that each of these three genes DMWD, DMPK, and SIX5 is independently regulated in mice and humans (Eriksson et al., 2000). 1.2.1 DMPK The DMPK gene encodes for a serine/ threonine protein kinase, which is expressed mainly in skeletal muscle, heart, stomach, testis and brain (reviewed by Groenen and Wieringa, 1998; Ueda et al., 2000). DMPK transcripts are subject to extensive cell type dependent alternative splicing. This alternate splicing is well conserved in mouse and man and results in 6 major splice forms (Groenen et al., 2000). These isoforms all contain the (CUG)n repeat in the 3'-UTR of the mRNA. A minor DMPK splice isoform, identified recently in human, is characterized by lack of a (CUG)n repeat, due to use of an alternative splice acceptor site in exon 15 (Tiscornia and Mahadevan, 2000). Alternative splicing in DMPK defines the presence or absence of the five amino acid motif val-ser-gly-gly-gly (VSGGG), and the nature of the C-terminus (Groenen and Wieringa, 1998; Groenen et al., 2000). The VSGGG-motif and different C-termini confer distinct properties to the six DMPK isoforms (Wansink et al., unpublished). Next to the two 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. alternatively spliced domains, all DMPK isoforms share an N-terminal leucine-rich domain, a ser/thr protein kinase domain and a coiled coil domain. The finding that DMPK transcripts bearing long (CUG) repeats in their 3’ UTR are retained in the nuclear foci of cells from myotonic dystrophy patients (Davis et al., 1997; Mankodi et al., 2001; Taneja et al., 1995) provided a stimulus to the understanding of the molecular pathogenesis of the disease. It has been demonstrated that the cytoplsmic levels of DMPK transcripts with long (CUG) repeats drop to almost undetectable levels (Krahe et al., 1995; Wang YH et al., 1995). Investigations to determine the role of DMPK \n DM1 pathology have been done using the Dmpk knock-out mice as disccussed in section 1.3.1. 1.2.2 DMWD The DMWD gene (formerly Dmr-N9 or gene 59 in mouse and human, respectively) is located 500 bp upstream of the DMPK gene as shown in Figure (1.1). A homology search conducted with the full length sequences of mouse and human shows that DMWD is evolutionary conserved with homologs of this gene being present in C. elegans (C08B6.7), S. pombe (YDE3), A. thaliana (T2N18.8), D. melanogaster (CG6420), and A. nidulans (CreC) (Wansink et al., 2003). DMWD is a member of a large family of eukaryotic WD-repeat containing proteins. Other members of this family include the (3-subunit of GTP-binding proteins 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. involved in neurological development, transcription, mRNA processing, vesicle transport and cytoskeletal assembly (Neer et al., 1994). DMWD is mainly expressed in the testis and the brain, with low levels of expression in many other tissues (Jansen et al., 1995; Eriksson et al., 2000). These observations, together with DMWD being a member of the protein family involved in a diverse range of cellular processes, suggest a possible role of DMWD in male sterility, testicular atrophy and mental retardation associated with DM1 (Jansen et al., 1995; Shaw et al., 1993). In DM1 patients with large repeats, it has been demonstrated that DMWD expression is equivalent to the wild type allele but in the cytoplasm a reduction off 20-50% was observed. There was an inverse correlation between (CTG) repeat expansion sizes with DMWD expression levels (Hamshere et al., 1997; Alwazzan et al., 1999; Eriksson et al., 1999; Frisch et al., 2001). Thus the next goal should be to define the repeat expansion thresholds that could be involved, and to develop animal models to study the pathogenic effects that result from decreased DMWD levels. 1.2.3 SIX5 The SIX5 gene (formerly known as DMAHP), which was first identified by Johnson and coworkers (Boucher et al., 1995), is located 3' of the DMPK gene as shown in Figure (1.1) and is the vertebrate homologue of sine oculis, a gene essential for the development of the visual system of Drosophila. SIX5 encodes a homeodomain transcription factor and is a 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. member of the SIX (sine oculis homeobox) family (Kawakami et al., 2000). Several different SIX5 transcription initiation sites have been identified. These sites are conserved between man and mouse, and are located downstream of the (CTG) repeat (Murakami et al., 1998). Expression of Six5 has been detected during the E7- E17 stages of mouse embryogenesis (Murakami et al., 1998). In adult tissues, SIX5 is mainly expressed in skeletal muscle, heart, eye, kidney, liver, lung and small intestine (Kawakami et al., 1996; Heath et al., 1997; Murakami et al., 1998; Ohto et al., 1998; Winchester et al., 1999; Eriksson et al., 2000). Remarkably, the expression patterns of SIX5 and DMPK are essentially overlapping, albeit that the SIX5 levels are generally 5-50 folds lower than those of DMPK (Heath et al., 1997; Eriksson et al., 2000). A screen for downstream targets of SIX5 revealed several candidate genes expressed in somites, skeletal muscle, brain and meninges, one of which was IgfbpS, encoding a component of IGF signaling pathway (Sato et al., 2002). In an independent study, the two DNA binding domains of Six5 were expressed as GST fusion proteins. It has been shown that these recombinant proteins bind to elements in the Na+ /K+ -ATPase a-1 subunit gene, but not to putative Six5 binding sites in the DMPK promoter (Harris et al., 2000). In Drosophila, the SIX5 homologue D-SIX4 was found to play a role in gonad and muscle development (Kirby et al., 2001). In conclusion, the presumed function of SIX5 in tissues involved in DM1 pathology 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (e.g. skeletal muscle, eyes, testis), and the finding that expansion of the (CTG) repeat decreases Six5 expression levels (discussed below) has opened the possibility that DM1 must be considered a multigenic disease. Evidence to support this hypothesis has been sought in the Six5{-/-) mouse strains (see section 1.3.2: Six5 knock-out mice). 1.3 Current approaches to dissect the DM1 molecular pathogenesis Understanding the molecular basis for the heterogeneity of the DM1 pathology, poses a significant challenge. The presence of (CTG) triplet repeat expansion in the mutant DMPK have been implicated in several molecular events that may account, independently, for the multisystemic aspects of this disease (Groenen et. al., 1998; Tapscott, 2001). Due to its position in the 3’ UTR, the (CTG) tract in the DMPK mRNA does not get translated. Thus defining the cellular events leading to DM1 pathology is complicated by the fact that mutant DMPK gene produces a normal DMPK protein. Therefore gain of function at the protein level is ruled out in this case. Thus, mechanisms of DM1 pathology may involve partial loss of function of DMPK and/or a neighboring gene, or a novel gain of function of the DMPK mRNA containing the repeat sequence. Currently three different mechanisms have been proposed to explain DM1 pathology. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.3.1 The DMPK haploinsufficiency model This model proposes that the expanded repeat in the DMPK transcript (present as a repetitive CUG sequence in the mRNA), alters the processing of the DMPK RNA thereby resulting in the deficiency of the normal protein. It has been established that the mutant DMPK transcript is retained in the nucleus, which prevents its transport to the cytoplasm where it would be translated into protein in the normal scenario. To test whether the deficiency of DMPK may lead to the progression of DM1 pathology, two groups created Dmpk knock-out mice by employing homologous recombination in embryonic stem cells. Only the mice carrying two defective copies of the gene (homozygous knock-out) developed a mild myopathy. Also, the Dmpk knock-out mice could not reproduce the eye and muscle defects that are characteristic of the DM1 disease (Jansen et al., 1996; Reddy et al., 1996). Analysis of the cardiac conduction defects in these mice demonstrated cardiac conduction defects that are similar to those found in human DM1 patients (Berul et al., 1999). Dmpk(-/-) mice developed first, second and third degree atrio-ventricular (AV) blocks. While the Dmpk{+/-) mice developed only first degreee AV blocks, which is strikingly similar to the cardiac conduction defects observed in DM1 patients. Thus the dosage alterations of Dmpk result in cardiac manifestations of DM1. Further it was established that the cardiac conduction defects are age-dependent and progressive as well (Berul et 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. al., 2000). The in vivo electrcardiography demonstrated higher grades of AV blocks with age and progressively prolonged PR interval in the Dmpk(-/-) and Dmpk(+i-) mice. Dmpk null mice were investigated in order to understand the mechanism of cognitive dysfunction in DM1 (Schulz et al., 2003). Dmpk is a member of two networks that are implicated in cytoskeletal dynamicity and synaptic plasticity. Dmpk null mice showed a normal synaptic physiology in the Hippocampal area and long-term synaptic potentiation (LTP). However, a significant decrease in decremental phase of long-term potentiation was observed which might be a reason for the cognitive dysfunctions. DMPK is speculated to have a role in the cross-talk between the adhesion-dependent and chemically stimulated transducing systems as they regulate the synaptic function and plasticity. 1.3.1 The Chromatin structure model This model speculates that an alteration of the local chromatin structure by (CTG) expansion, leads to transcriptional repression of the adjacent transcription factor SIX5 gene and a corresponding reduction in SIX5 expression (Wang et al., 1994; Otten and Tapscott, 1995). Several studies have showed the decreased expression of the transcription of the DMWD (formerly gene 59), which is located 5’ of DMPK and SIX5 found in 3’ UTR of DMPK in some DM1 patients (Klesert et al., 1997; Thornton et al., 1997; Korade-Mirnics et al., 1999). However it is important to note that this may be due to the inhibition of mRNA nucleo-cytoplasmic transport rather 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. than a transcriptional defect (Alwazzan et al., 1999). Since DMWD gene is highly expressed in testis and brain, its transcriptional down regulation may be implicated in the testicular atrophy and cognitive disturbances that are associated with DM1. The SIX5 family of proteins is particularly interesting because they have been implicated in the regulation of muscle cell differentiation and sodium ion homeostasis, both of which are disrupted in DM1. To investigate if SIX5 dosage effects could produce the DM1 phenotype, Six5 knock-out mice were made independently by two labs (Klesert et al., 2000; Sarkar et al., 2000). Both Six5(-/~) mice lines developed ocular cataracts at a rate higher than the wild type animals. In one of the mice line, Six5{+/~) mice had significantly enhanced cataracts formation (Sarkar et al.,2000). Thus the formation of cataracts in mice with either one or both defective copies of Six5 gene suggests that the cataracts in human DM1 might result from decreased expression of Six5 (Klesert et al., 2000; Sarkar et al., 2000). Though Six5 is expressed in (developing) skeletal muscle and is involved in the muscle development, no abnormal skeletal function was found in Six5 knock-out mice. Also, Six5(+/-) mice did not show Na+ channel gating abnormality of Dmpk(+/-) mice (Mistry et al., 2001). Wakimoto et al., (2002) studied the cardiac phenotype of DM1 in Six5(+/-) mice. Prolonged QRS duration and the conduction delays characteristic of the initial phenotypes of adult onset cardiac conduction abnormalities in DM1 patients were detected in Six5 heterozygous mice. 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thus the loss of Six5 protein contributes to cardiac conduction disturbance in mice and is associated (either directly or secondary to Dmpk downregulation) with the AV-conduction defects. 1.3.3 The dominant RNA model This model proposes that the triplet repeat expansion in the DMPK RNA interacts with RNA binding proteins blocking their normal activity (termed as a toxic gain-of-function) and thereby results in the disruption of the cellular metabolism (Taneja et al., 1995; Wang J et al., 1995; Davis et al., 1997; Hamshere et al., 1997). Recent studies using transgenic mice and C2C12 myoblast culture systems provide critical data that support this hypothesis (Mankondi et al., 2000; Amack et al., 1999). The expression of RNAs containing about 250 (CUG) repeats produces myopathy and myotonia as characteristic of DM1 in mice (Mankondi et al., 2000). Since the repeat was placed in the 3’ non-coding region of skeletal actin transgene which is unrelated to Dmpk gene, the pathology was independent of the context and specific to (CUG)n repeats. Thus it was concluded that it is the (CUG) repeat expansion in the RNA that is responsible for the myopathy. Similarly, expression of different transgenes containing the (CUG) repeat expansion inhibits differentiation of cultured muscle cells. Thus these studies conclude that the expanded (CUG) repeat in the non-coding region of RNA has a toxic gain-of-function effect. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The mechanism of the RNA gain-of-function is believed to occur due to the abnormal binding of different RNA-binding proteins to the (CUG)n repeat tracts or by the maldistribution of the transcription factors essential for various cellular processes. Several such RNA binding proteins have been identified and include proteins such as (CUG) repeat binding protein (CUG-BP) and muscleblind (MBNL) (Lu et al., 1999; Miller et al., 2000; Timchneko et al., 1996). It has been shown that RNA containing more than 20 repeats forms a double stranded structure with U/U mismatches and binds RNA binding proteins (Michalowski et al., 1999; Napieraa and Krzyosiak, 1997; Miller et al., 20000; Tian et al., 2001). One of these proteins, MBNL (EXP family), is a zinc -finger protein, which exhibits striking sequence specific binding to double stranded (CUG) repeats. Though the function of this protein is not known completely, MBNL protein is mammalian homologue of the Drosophila muscleblind protein. In Drosophila, the absence of muscleblind leads to eye degeneration and defects in terminal muscle differentiation (Begemann et al., 1997; Artero et al., 1998). MBNL has been found to co-localize with the expanded repeats in the nuclear foci in DM1 patients and skeletal muscle tissue (Fardaei et al., 2001and 2002; Mankondi et al., 2001; Miller et al., 2000). CUG-BP and ETR-3 are members of the CELF family of proteins that are involved in regulation of splicing of different mRNAs by binding to conserved intronic elements containing U/G rich motifs (Charlet-B et al., 2002; Ladd et al., 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2001). In contrast to MBNL, CUG-BP does not bind double stranded RNA containing (CUG) repeat expansion and does not co-localize with the DM1 foci (Fardaei et al., 2001; Michalowski et al., 1999). It has been found that CUG-BP binds to the base of the stem loop structure formed by (CUG) RNA (Michalowski et al., 1999). However, the expression of transcripts containing (CUG) repeat expansion in COS cells results in the increase in the steady state levels of CUG-BP (Timchenko et al., 2001). The mutant RNA in DM1-affected cells was found to bind and sequester transcription factors (TFs) and thus leading to around 90% reduction of selected TFs from active chromatin (Ebralidze et al., 2004). A model of Myo-D generated myocytes from normal and DM1 patients was used to obtain equivalent muscle specific DMPK gene induction in control and mutant cells. The DM1 cells showed that the mutant RNA (C TG )ioo co-precipitated with different transcription factors. These transcription factors represented the three classes, associated normally with cell maintenance, activation and differentiation. Following a period of four weeks after Myo-D induction DM1 affected cells showed maldistribution of various TFs between active chromatin and RNP complexes. Eventually RNP became the dominant site for these TFs. This maldistribution (leaching) of TFs lead to reduced expression of diverse genes like ion transporter CIC-1 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (1.2): Myotonic Dystrophy: molecules and signs W S B B M Molecule change yotonic dystrophy: Molecules 8 Related features ( iM P * Disease Mechanism Dmpk Muscle weakness Cardiac conduction A Na+ channel defects Potentiation A in hippocampal- area Cataracts Cardiac conduction defects Mvotonia Reduced Protein from mutated gene Reduced Protein from neighboring gene reduction Six5 reduction RNA with Toxicity of long CUG repeat RNA more Insulin resistance CUG repeats Cl' channel defects Reference: http://www.neuro.wustl.edu/neuromuscular/musdist/pe-om.html Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that has been implicated in myotonia. However, the overexpression of TF specificity protein 1 (Sp 1) in DM1-affected cells restored the low levels of CIC-1 mRNA to normal. The exact mechanism by which the RNA gain-of- function lead to the pathology remains to be investigated. The outcome of all the three mechanisms may be summarized in Table (1.2). Thus all the three models contribute to DM1. The extent to which each mechanism contributes to the clinical features of DM1 is not known completely. It is interesting to note that each feature cannot be traced to one single effect. In conclusion, together with the transgenic mouse model consisting of human skeletal actin gene containing (CTG) repeat expansion (showing myotonia and muscle abnormalities), the Dm 15 knock-out model (with cardiac conduction defects and mild myopathy) and the Six5 knock out model (with cataracts) suggest that DM1 is a multimechanism disease involving multiple genes. 1.4 DM2- A Parallel universe Soon after the development of (CTG) repeat diagnostics, a category of patients with a myotonic dystrophy phenotype similar to DM1 and not carrying triplet repeat expansion in DMPK gene as in DM1 was identified (reviewed in IDMC, 2000, Meola, 2000; Ranum and Day, 2002). The mutation in patients with this form of DM, designated as DM2 or PROMM, (MIM 6026668) was mapped to chromosome 3q21.3 (Ranum et al., 1998) 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and recently has been characterized as an expanding (CCTG) repeat (Liquori et al., 2001). The DM2 families have myotonic myopathy, cataracts, cardiac conduction defects. Thus both DM1 and DM2 have repeat sequence C(CUG) as a cause of the pathogenesis. Thus expression of the (CUG)n repeat may contribute to the common features of these two diseases. Mouse models, which would test this hypothesis, are yet to be developed. Table (1.3) compares the clinical symptoms of two forms of Myotonic Dystrophy. 1.5 Hypothesis The presence of single mutation in the 3’ untranslated region of the DMPK gene and the involvement of the multiple systems in DM1 patients make the understanding of DM1 pathology a challenge. On reviewing the three mechanisms proposed to underlie the DM1 pathology, it can be concluded that each of them contributes in part to the characteristic phenotype of DM1. This view is supported by the appearance of partial DM1-like phenotype in various mouse models studied to date. As given in Table (1.2), it has been reported that the Dmpk knock-out mice shows muscle weakness and myotonia is seen in mice overexpressing (CTG) repeat. But there are no reports till date to suggest if Six5 knock-out mice and (CTG)n overexpression leads to muscle weakness. Thus in the present 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (1.3) DM1 vs. DM2: comparative features DM 1 vs DM 2 (PROMM) Sian i DM1 I DM2 Epidemiology ! Widespread j European Onset Age j 0 to Adult | 8 to 60 years Anticipation | + | Mild Weakness Face Ptosis Proximal Distal Sternomastoid + I Mild + ! Mild + I Mild + j + + j Variable Muscle pain Calf hypertrophy I + ! + Cataracts Balding + | + + I + Cardiac arrhythmias Gonadal failure + + " + ' ........20%......... s Hyperglycemia Hypersomnia + | 20% + [ Variable Hyperhidrosis Cognitive disorder Variable | + Mild to Severe j Mild Cogenital form EMG: Myotonia + I + ! + Chromosome Mutated gene 19q13.3 3q21 DMPK j ZNF9 Mutation type Repeat size (CTG) repeats ](CCTG) repeats 100 to 4,000 | Mean-5,000 Reference:http://www.neuro.wustl.edu/neuromuscular/musdist/pe-eom.html Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. study I hypothesized that Six5 and (CTG) repeat overexpression also lead to muscle weakness. A 2-3-folds reduction in the expression of Six5, a gene immediately downstream of Dmpk led to further investigations on Six5 knock-out mice. The analysis of these mice showed that Six5 knock-down could recapitulate some of the features of DM1 phenotype (Klesert et al., 2000; Sarkar et al., 2000). Heterozygous loss of Six5 in mice has previously been shown to cause ocular cataracts, however it’s role in myotonia and muscle weakness has not been investigated. This study was aimed to test if there are any muscle abnormalities (myotonia or reduced force generation) in these Six5 knock-out mice. The Six5 knock-out mice created by Sarkar et al., (2000) have been used to investigate muscle electrophysiology resulting from Six5 knock-out. Previous studies by Mankondi et al., (2000) have shown that the ectopic expression of an a-actin cassette containing a 0.7 kb tract of (CTG) repeats in skeletal muscle leads to muscle myotonia, fiber loss and formation of central nuclei. However the present study aims at understanding the role of (CUG) repeat length and dosage in muscle wasting and reduced force generation. For this, mice created in Reddy laboratory with different length and dosage of (CTG) repeats have been used to analyze the muscle function. 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2 MATERIALS AND METHODS 2.1 Six5 knock-out mice 2.1.1 Strategy to knock-out Six5 gene Reddy lab had previously created Six5 knock-out mice to investigate the second proposed mechanism, “The chromatin structure model” for DM1 pathology. Figure (2.1) explains the knock-out plasmid used to down regulate the gene via homologous recombination Sarkar et al. (2000). The targeting strategy replaced 398 bp upstream of the start codon, Six5 coding sequence, and approximately 180 bp downstream of the termination codon with a PGK-neo cassette in the same transcription orientation as the Six5. 2.1.2 Screening of the Six5 knock-out mice Following the creation of these two mice lines, Southern blots were used to screen the founder mice. By Southern blots, Six5 knock-out homozygous, heterozygous and wild type mice were identified. Figure (2.2) shows the genotyping strategy adopted for the screening of founder Six5 knock-out mice. The 700 bp probe detected a band of 12.4 kb in the mutated allele due to mutation of the EcoR-1 restriction site in the middle. In the wild type mice, the probe detected a band of 6.3 kb because the middle Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a wild-type allele Dm 16 S»*S Rl Rl 8 m B g < I * ^ | iff _ i. nTiMr^- l P l l ® p i i ■ ■ 11 n M i m i Q i a n I' £ 6.3 kb P r o b e l o r S o u i h o r r i targeting R| ^ m Bel Bm ^Bgl oonstruot , m l2 J4^ s| f ______ |-| iisv-nf~l— c targeted „ „ allele Dm 15 Bm Rl Is 9 | 1 0 1 2 t h "Ifei.Rf B V B , L * * w I ■ ■■ I I I 11 iiK s a m 12.4 kb f Probe for ^ Southern Targeted inactivation of mouse Six5. a, Map of the wild-type mouse Dm 15 locus. Dm 15 exons 8 -1 5 are shown as shaded boxes. The (CTG) expansion is located in D m 15 exon 15. The three Six5 exons are designated as EA, EB and EC. b, Map of the Six5 targeting construct. The mutated EcoRI site is shown as Rl*. c, Map of the mutated D m 15 locus. All three Six5 exons are replaced by a PGK-neo cassette. Bm, BamHI; Rl, EcoRI; Bgl; Bgr/ll; Rl*, mutated EcoRI site. The probe used for Southern-blot analysis of the wild-type and the mutated Dm 15 locus is underlined. Figure (2.1) A schematic Diagram to show the Six5 knock-out construct. Reference: Sarkar et al., Nature Genetics, 2000 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EcoR-1 digestion site gave a smaller DNA fragment corresponding to this region of genomic DNA. Therefore 6.3 kb band is seen in the homozygous knock-out mice and 12.4 kb band is seen in the wild type mice. For the heterozygous mice, both 6.3 kb and 12.4 kb bands are seen. The (3-actin gene was used as a control for mRNA quality and quantity. The genomic DNA from these mice was used as control sample for the subsequent PCR genotyping. 2.2 a-actin (CTG) repeat overexpresser mice 2.2.1 a-actin (CTG) repeat length plasmid A novel strain of mice expressing (CTG) repeat sequence specifically in the skeletal muscle was developed in Reddy laboratory. Figure (2.3) shows the diagrammatic representation of the plasmid used to generate two mice lines carrying different lengths of the repeat where “n” equals 700 bp for (CTG)7 o o and 900 bp for (CTG)g0 o . a-actin promoter was utilized to drive the Lac-Z- (CTG) repeat overexpression, a-actin promoter is specifically expressed in the skeletal muscle. Thus the transgene was designed such that the expression of (CTG) repeat is specific to skeletal muscle. A 800 bp (3-intron sequence was inserted between the promoter and the [3 - galactosidase gene. The presence of the intronic sequence between promoter and the coding region of the gene provides a better spatial arrangement for binding of the transcription factors on the chromatin. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ii ~ i ~ ! “ + / “ t/t f/i- | w ild -typ e allele • 12.4 kb m utant allele - 6.3 kh I w T ™ \ 1 e heart brain muscle eye H 1 2 3 4 5 6 7 8 0 10 1 1 1 2 1.1 kb ■ r J M 1 Z 3 4 S 6 ! a 9 10 1 1 IjT] 0.46 k b - Targeted inactivation of mouse Six5. (d) Southern-blot analysis of tail clip DNA from wild-type, Six5 + / - and Six5 - / - mice, digested with EcoRI and probed with a 0.7 kb Bglll/EcoRI fragment, demonstrates that Six5 - / - mice lack the wild-type 6.3 kb EcoRI fragment. ( e,f) R T -P C R analysis of total RNA isolated from tissues derived from wildtype, Six5 + /- and Six5 - / - mice demonstrates the absence of Six5 transcripts from Six5 - / - mice. Tissues examined include heart (lanes 1-3 ), brain (lanes 4 -6 ), muscle (lanes 7 -9 ) and eye (lanes 1 0 -1 2 ). Lanes 1,4,7,10 show wild-type tissue; lanes 2,5,8,11 show Six5 + /- tissue; lanes 3,6,9,12 show Six5 - / - tissue. M, 1 kb DNA ladder, f, R T -P C R analysis of the mouse p-actin gene was used as a control for m RNA quality and quantity. Figure (2.2) Six5 genotyping by Southern blots. (Reference. Sarkar et al, Nature Genetics, 2000) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (CTG). BamHI Hfnd H i TSS Stop Sfil Sfil ATG P-Galactosidase BGH p-globin intron Figure (2.3) Ectopic expression of (CTG) repeats in skeletal muscle (PS Sarkar, personal communications) Approximate length of different fragments: a-actin promoter (2.2 kb), intron- 800 bp, yff-galactosidase gene (3.1 kb), (CTG) repeat length (700 /900 bp), BGH poly A (230 bp). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. An extra sequence of BGH poly A (230 bp app.) was added at the 3’ end of (3-galactosidase gene. The addition of poly-A sequence facilitates the nucleo-cytoplasmic transport of the transcript. The repeat sequence was cloned between the 3’ UTR of (3-galactosidase gene and the BGH polyA sequence. The transgene was inserted into the multiple cloning site (MCS) of pBluescript pKS (+/-) as the backbone plasmid. 2 .2 .2 Identification of the mice carrying a-actin (C T G )7oo/(CTG )g0o repeat length plasmid Mice were screened for (CTG )7oo/(CTG )g0o overexpression using the Northern blots that depicted the length and extent of expression in the transgenic mice. The transgenic mice expressing 700 bp repeat expansion was found to show higher levels of expression as compared to mice expressing 900 bp repeat length. These expression levels corresponded to the signal from (3-galactosidase activity on the Northern blot. The following generations from founder (C T G )7oo/(CTG)9oo were maintained and used for the present study. 2.3 Genotyping the mice lines by PCR amplification Genomic DNA was obtained from these mice to PCR amplify the gene of interest. Genomic DNA was extraction was done using the following protocol. (Molecular cloning: a laboratory manual by Maniatis et al. 1989). 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stock solutions (sterilized) Lysis buffer (100 mM Tris-CI, pH =8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCI) Proteinase K (10 mg/ml) Tris treated phenol: chloroform: isoamyl (25: 24: 1, v/v) Choloroform 100% isopropyl alcohol TE buffer 70% ethanol Genomic DNA extraction and purification ❖ About 1 cm of mice tail was cut and put it into the micro-centrifuge tube. ❖ 600 /j \ of lysis buffer with 100 yt/g/ml of proteinase K was added to the tube containing the mice tail. ❖ The sample was incubated at 55°C with little agitation overnight. ❖ The tubes were taken out and 600 fj\ of Tris-treated phenol was added to each tube. The tubes were inverted several times to mix the solutions thoroughly. ❖ The tubes were centrifuged at 14,000 rpm for 10 min, at 4°C. ❖ The top aqueous layer (containing the DNA) was transferred to a new tube. Same volume of Tris-treated phenol solution was added to the tube. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ❖ Step 5 was repeated once more. ❖ The top layer was transferred to a new tube. Same volume of choloroform was added and gently mixed. ❖ Step 5 was repeated once more. ❖ Again the top aqueous layer was transferred to a new tube. Same volume of isopropyl alcohol (100%) was added to the sample. ❖ The tubes were inverted several times to mix the solution or until formation of DNA strings was observed in the tube. ❖ The samples were incubated at -8 0 C freezer for 20 minutes. ❖ The pellet was centrifuged at 14,000 rpm for 10 minutes at 4°C. ❖ The alcohol was poured off and the pellet was washed with 500 fj\ of 70% ethanol. ❖ The pellet was air dried for about 10 minutes and the DNA pellet was resuspended in 300//I of Tris- EDTA buffer. 2.3.1 Strategy for genotyping the Six5 knock-out mice Genotyping of Six5 knock-out was based on amplification of Six5 gene and the neomycin gene. Since wild type mouse has both copies of Six5 intact, it should amplify only Six5. On the other hand the homozygous knock-out mouse has both the copies of Six5 removed, so it should not give any amplification for Six5 but rather should amplify the neomycin gene. Since heterozygous mouse has one copy each of Six5 and neomycin, it 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. should amplify both the Six5 and neomycin genes. Table (2.1) depicts the strategy for genotyping Six5 mice. Six5 amplification Primer design: I designed primers and custom ordered them from Invitrogen to amplify certain region of the Six5 gene. They amplified a region of 291 bp. The lengths of both forward and reverse primers were 21 base pairs. The sequence of the forward as well as the reverse primers was as follows: Forward (5’ to 3’): ACT ACG GAG GAT GAG TCC AGC Reverse (5’ to 3’): GCA CTT CCT CCT GTG AGT AGC Thermal cycling The PCR reagents were optimized to obtain required PCR product as in Table (2.2). The 10X PCR buffer, Taq DNA polymerase, MgCh and dNTPs were all ordered from Invitrogen. The PCR was conducted in PTC- 200, Peltier Thermal Cylcer (MJ research, USA). Thermal cycling was carried as follows: ❖ Primary denaturation at 95 °C for 5 minutes. ❖ Secondary denaturation at 94 °C for 1 minute. ❖ Annealing at 56°C for 1 minute. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (2.1): Genotyping of Six5 knock-out by PCR amplification Genotype PCR product Wild type heterozygous homozygous (+/+) (+/-) (-/-) Six5 Neomycin Wildtype mice amplify a band for the two intact copies of Six5, Homozygous mice amplify a band for neomycin that replaced both the copies of Six5. Heterozygous mice amplify one band for the intact copy of Six5 and one for neomycin that replaced second copy of Six5. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ❖ Extension at 72 °C for 1 minute. 30 cycles of secondary denaturation to extension ❖ Final extension at 72 °C for 10 min Neomycin gene amplification Primer design: Primers were designed previously in the lab and custom ordered from Invitrogen to amplify certain region of the neomycin gene. They amplified a region of 315 bp. The lengths of both forward and reverse primers were 21 base pairs. The sequence of the forward as well as the reverse primers was as follows: Forward (5’ to 3’): CGC ATT GCA TCA GCC ATG ATG G Reverse (5’ to 3’): GGA GAG GCT ATT CGG CTA TGA C PCR reaction ratios were same as those used for the Six5 amplification. Thermal cycling was carried as follows: ❖ Primary denaturation at 95 °C for 5 minutes. ❖ Secondary denaturation at 94°C for 1 minute. ❖ Annealing at 62°C for 1 minute. ❖ Extension at 72°C for 1 minute. 30 cycles of secondary denaturation to extension ❖ Final extension at 72°C for 10 minutes Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (2.2) Six5 amplification: PCR reagents, volume and Concentration Reagents Volume Final Concentration 10X PCR Buffer 3 pi 1X 50 mM MgCI2 1.5 pi 1.5 mM 10 mM dNTP mixture 1.00 pi 0.5 mM each Forward primer 1.00 pi 0.2 pM Reverse primer 1.00 pi 0.2 pM Taq DNA polymerase (recombinant) 0.5 pi 2.5 U Genomic DNA (25 ng) X Total (add sterile water) 30 pi - (Molecular cloning: A laboratory manual by Maniatis et al, 1989) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3.2 Genotyping (C T G )7oo /(C T G )90o overexpresser mice Once the founder mice for (C T G )7oo and (CTG)goo were screened, their litter were used for further investigations. Subsequent generations of these transgenic mice were genotyped by amplifying the Lac-Z gene which would be expressed only in the (C T G ) repeat expressing mice, by virtue of the transgene design. Lac-Z gene amplification Primer design: Primers were designed previously in the laboratory and custom ordered from Invitrogen to amplify certain region of the Lac-Z gene. The lengths of both forward and reverse primers were 21 base pairs. The sequence of the forward as well as the reverse primers was as follows: Forward (5’ to 3’): GCG TTA CCC AAC TTA ATC GCC Reverse (5’ to 3’): TGC ACC ATC GTC TGC TCA TCC They amplified a region of approximately 1.15 kb. PCR reaction ratios were same as those used for the Six5 amplification (Table 2.2 ). Thermal cycling was carried as follows: ❖ Primary denaturation at 95 °C for 5 minutes. ❖ Secondary denaturation at 94°C for 1 minute. ❖ Annealing at 48°C for 45 seconds minute. ❖ Extension at 72°C for 2 minute. 30 cycles of secondary denaturation to extension ❖ Final extension at 72 °C for 8 minutes 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.4 Muscle electrophysiology 2.4.1 Muscle sample preparation In vivo muscle preparation was chosen for electromyography (EMG). Animals were anaesthetized by I.P. administration of 80/10 mg/kg of ketamine /xylazine. The animal was weighed and the required patch of skin was shaved off. The skin was cut open to expose the muscles to be used for the EMG recordings. In vitro muscle preparation: the same animal after the recording of in vivo EMG was euthanised by I.P. injection of 100 mg/kg Nembutal. Soleus and Extensor Digitorum longus (EDL) muscles were isolated and removed in entirety for other electrophysiology studies. The whole process of muscle preparation was carried out with adequate oxygenation of the Kreb’s solution in the dissection chamber. Table (2.3) gives the chemical composition of the Kreb’s solution. After dissection, the muscle was suspended in Kreb’s solution maintained at 27°C. 2.4.2 Electrophysiological investigations To determine if myotonia or muscle function abnormalities were present in the Six5 knock-out mice, (CTG)7oo and (C T G )90o transgenic mice, I performed electromyography and measured muscle contractile properties . All these data acquisition and processing was done in conjunction with Dr Personius and with his instrumental set-up at SUNY Buffalo. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Electromyography Following skin preparation and muscle visualization in the K/X anaesthetized mice, EMG was performed. Few of the muscles tested for myotonia were Soleus, EDL, paraspinal, and intrinsic muscles of hind paw. But the main focus was on Soleus and EDL, for which the muscle contractile properties were also studied. Myotonic runs were defined as trains of action potential waxing and waning in amplitude and frequency lasting longer than 1 sec. A 30 gauge concentric needle (Medtronic) was chosen as electrode and was inserted into each muscle 4-5 times. A series of EMG tracing were filtered (500 Hz high pass) and differentially amplified (TDT digital bioamplifiers, DT Technologies). Signals were acquired and analyzed by Spike2 (CED, Inc). Muscle contractile properties The soleus and EDL muscles were isolated and removed from the mice as per the method described in section 2.3.1. Silk suture was tied to each tendon of the isolated muscle. The muscles were placed in an experimental chamber filled with Kreb’s solution (chemical composition given in Table 2.3), adjusted to pH=7.4. The chamber was perfused continuously with 95 % 0 2 -5% C 02 and maintained at a temperature of 27 °C. One end of the muscle was attached to the lever arm of a force transducer (model 300B, Aurora Scientific Inc.). The other end of the muscle was tied to a steel hook of a micromanipulator. Contractile 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (2.3) Kreb’s solution: chemical composition Chemical Concentration (mM) Quantity (for 1 liter) Sodium chloride 137 8.0 g Potassium chloride 4 298 mg Magnesium chloride 1 203 mg Sodium pot. Monobasic 1 138 mg Sodium bicarbonate 12 1.01 g Calcium chloride 2 294 mg D- glucose (anhydrous) 6.5 1.17 g 3 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. properties were measured on EDL and soleus muscles in vitro using techniques described previously (Lewis et al., 1986, Aurora Scientific Inc. 300B lever systems). The muscles were stimulated by an electric field generated between two silver electrodes placed longitudinally on either side of the muscle. I determined optimal muscle length (L0), peak twitch force (Pt), time to peak twitch tension (TPT), time for tension to decay from maximum to half-maximum twitch tension (1/2RT), and maximal contraction force (P0). To assess the extent of muscular fatigue, we stimulated the muscles with 350 ms trains at 1 Hz with a pulse frequency of 35 Hz and 0.2 ms duration for ten minutes (Masters 8 stimulator, AMPI). A fatigue index was calculated by dividing the contractile force produced at ten minutes by the initial contractile force. A frequency-force curve was established at frequencies of 5, 10, 20, 35, 50, 65, 80, 100, 150 and 200 Hz for soleus and EDL muscles. The absolute P0 was determined from the plateau of the frequency-force relationship. 2.4.3 Data Analysis Following formulae were used for calculations: Grams force =(voltage *60.4) + 0.121 Newtons =(grams force /1000) *9.81 Muscle volume (cm3) = Muscle weight (g) /Muscle density (g /cm3 ) Muscle cross-sectional area (cm2 ) = Muscle volume (cm3 ) /L0 (cm) Muscle density = 1.056 (g /cm3 ) 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Given below are the different parameters that were measured in this section of study. These parameters are explained further in “Chapter 3: Results”. L0= optimum length at which maximum twitch force is obtained P0 = maximal tentanic force (N/cm2 ) TPT = time to maximal force of muscle twitch (ms) 1/2RT = time from maximal force to !£ of maximal force during twitch (relaxation time, ms) FI = ratio of force produced at 10 min / initial force (fatigue index) Twitch = maximal force of muscle twitch (N/cm2 ) Statistical analysis: Sigmastat was utilized for the analysis of difference in the mean values of muscle contractile parameter among the different groups of animals. 2.5 Muscle histology 2.5.1 Muscle sample preparation The age-matched animals from Six5 knock-out line, in all three genotypes were selected for histological studies. The experimental animals were sacrificed and two muscles namely: Soleus and EDL were dissected and washed with phosphate buffer saline (PBS, pH =7.5). Tissues were fixed by sequential dehydration with increasing concentrations of ethanol. The outline for the process is as following: 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ❖ Overnight 4% para-formaldehyde (PFA) treatment at 4 °C ❖ PBS wash ❖ 50% ethanol treatment, for 30 minutes ❖ 70% ethanol treatment, overnight ❖ 90% ethanol treatment, for 30 minutes ❖ 100% ethanol treatment, for 30 minutes ❖ 100% ethanol treatment, for 30 minutes ❖ xylene treatment for 15 minutes at 65 °C ❖ 1:1 xylene and paraffin for 30 minutes ❖ Paraffin, overnight The next day paraffin blocks were prepared for these tissue samples in plastic molds (Fisher, USA). The paraffin blocks were allowed to solidify. Sections of 10 pm thickness were cut from these paraffin blocks. The wrinkles from the paraffin ribbons were removed by spreading them on 45°C water bath. The spread out sections were collected on glass slides (VWR corporation) pre-coated with lysine. The slides were allowed to air dry at room temperature. These sections were subsequently used for histological studies. 2.5.2 Hematoxylin and eosin staining Histological sections prepared previously were processed by regular H/E staining protocol as outlined below. Before staining, the paraffin had to be removed and the sample rehydrated. 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ❖ Xylene (solves the paraffin) 5 min (repeated one more time) ❖ 100% ethanol (solves the Xylene) 2 min (repeated one more time) ❖ 90% ethanol 2 min ❖ 70% ethanol 2 min ❖ Water rinse for 2 min (repeated one more time) ❖ Hematoxylin staining 1 and 1/2 minute ❖ Water rinse for a few times ❖ Dipped in ammonia water ❖ Water rinse again a few times After the staining process, the sample had to be dehydrated again before it was enclosed: ❖ 70% ethanol 2 min ❖ Eosin staining for 45 sec. ❖ Dipped in 95% ethanol a couple of times to remove excess stain ❖ 100% ethanol 5 min (repeated one more time) ❖ Xylene 5 min (repeated one more time) ❖ Mounting of samples The final fixing of the tissue was obtained by enclosing the samples using a resin between the glasses. Using DPX as the mounting resin, cover slip was placed carefully to cover the stained section of the tissue. Following the mounting, slides were allowed to dry at room temperature. 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3 RESULTS 3.1 Genotyping and Experimental set-up 3.1.1 Six5 knock-out mice Six5 PCR amplification The sequence of the forward as well as the reverse primers was as given. The primers annealed to the highlighted region of the gene as shown in Table (3.1). A product of 291 bp was obtained for the PCR amplification. Forward (5’ to 3’): ACT ACG GAG GAT GAG TCC AGC Reverse (5’ to 3’): GCA CTT CCT CCT GTG AGT AGC Neomycin PCR amplification: The sequence of the forward as well as the reverse primers was as given: These primers annealed to the highlighted region of the gene as shown in Table (3.2). A product of 315 bp was obtained by PCR amplification. Forward (5’ to 3’): CGC ATT GCA TCA GCC ATG ATG G Reverse (5’ to 3’): GGA GAG GCT ATT CGG CTA TGA C As explained earlier, wild type mice amplified only for Six5 gene, homozygous mice amplified only Neomycin gene, and heterozygous showed amplification for both Six5 and Neomycin genes. Figures (3.1) and 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.1 Six5 sequence (Source: http//www.ensemble.org) GCGGCGGCGGACTCTGGGTCCGCGAGTGGCCCGGGGTCTCCCCGGGAGACCGTGACCGA GGTGCC GACT GGCCTTCGCTTCTCGCCC GAAC AGGTG GC ATGCGT GTGCGAGGCGCTG CT GCAGGCGGGCCACGCCGGCCGCTTGAGCCGCTTCCTGGGCGCGCTGCCCCCGGCCGAGC GCCrACGTGGCAGCGATCCGGTGCTGCGCGCGCGGGCCCTAGTGGCCTTCCAGCGGGGT GAATACGCCGAGCTCTACCAACTTCTCGAGAGCCGCCCTTTCCCCGCCGCCCACCACGCCT TCCTGCAGGACCTCTACCTGCGCGCGCGCTACCACGAGGCCGAGCGGGCCCGTGGCCGT GCGCTGGGCGCTGTGGACAAATACCGGCTGCGCAAGAAGTTCCCTCTGCCCAAGACCATCT GGGATGGCGAGGAGACCGTCTATTGCTTCAAGGAGCGCTCGCGAGCGGCGCTCAAGGCCT GCTACCGCGGCAACCGCTATCCCACGCCTGACGAGAAGCGCCGCCTGGCCACGCTCACCG GCCTCTCGCTTACACAGGTCAGCAACTGGTTCAAGAACCGGCGACAGCGCGACCGCACTG GGACCGGCGGTGGAGCGCCTTGCAAAAGCGAG7CTOAr£5GeAACCCGflCraC0fi4fiF®*T ©AGTO^GGCGCA G71CCA GA GGACCTGGA GA GG GGTGTGG CCTiCCA TOG C7GCTGA GG C CCCCGCCCAGAGTTCCATCTTCCTGGCGGGGGCCACCTCTCCTGCAACGTGCCCTGCCTC CTCCTCTATCCTAGTGAATGGGAGCTTCCTGGCCGCCAGCAGTCCCCCAGCAGTGCTCCT CAATGGTAGCCCAGTCATTATCAATAGCTTGGCCCTAGGAGAGAACTCCAGCTTGGGGCC CTTmT^m ^g^Mm 0^CCCTCAACCACAGC(^AGTCTCCAAQG&3TCAGTGA GGCCAAGAA TFCTVfGGTGGTGGA CCCTCA GACAGGAGA GGTTCGACTGGATGAGG CTCA GTCTGA GGCCCCTGA GA CCAAA GGGGTTCA TGGGACTA CTGGA GA GGAAA TCCGA GGA G CCCTGCCCCAA GTAGTCCCA GGCCCCCCA GCTGCCTGCA CGTTTCGTGTGA CCCCGGGA G CTGTGCCrGCTGTGGCTGCTGGTCAQGTTGTACCGCTCTCCCCTTCTTCTGGGTACCCAAC AGGCCTGA GCCCCACCTCCCCACGGCTGAA CTTGCCGGA GGTGGTGCGCAQCTCTCA GGT GGTAACCCTGCCTCAGGCTGTGGGGCCACTCCAGCTGTTGGCAGCTGGGCCAGGCAGTC CTGTGAAGGTGGGA GCTGCAGCGGGGCCTA CCA A TGTGCA CCTGA TAAACTCTAGTGTGG GA GTGA CTGCGCTGCAA CTTCCCTCGTCCA C TGCTCCA GG AAACTTCCTCCTGGC C AACCC TGTGTCTGGT AGCCCCATTGTC ACTGGGGTAGCTGT GCAGCAGGGCAAGATCATCCTCACT GCCACCTT CCCCACCAGCATGCT CGTCTCCC AGGTCCTGCCTCCTGCCCCCAGTCTAGCCC TGCCCCT GAAGC AAGAGCC AGCT ATC ACAGTGCCT GAAGGAGCT CT CGC AGTGGGCCCCA GCCCCACCCT CCCAGAGGGT CACACT CT GGGGCCAATCT CTACTCAGCCACTGCCACCTGC TT CT GTTGTC AC CTCTGGC ACCAGCCT GCCTTTCT CCCCGGACTCCT CT GGCCTGCTTTCC A GCTTCTCAGCACCCCTACCTGAAGGTCTGATGTTGTCACCTGCAGCTGTGCCAGTCTGGCC AGCAGGGCTGGAACTGAGCACAGGAGTAGAAGGGCTGGGGACACAGGCCAOCCACACTGT GCTGAGGCTGCCAGACCCTGACCCCCAGGGACTGCTTCTGGGGGCTACAACAGGGACTGA GGTTGATG AGGGGCT AGAAGCTGAGGCCAAGGTCCTGACCCAGCTGCAGTCGGT ACCCGT GGAGGAGCCCTTGGAACTGTGACTGCCTGCATTTAACCACTTCTTCTGACAATGGTGTCAAG GTGCTAGGACAGGAAGAGGAACCCTCAGTAAAGTGGCTGCAGTCTGAAGTCCCACACTACA GCTCT CCT ACCT GGATAAGCT ATAAGCCCTAC AGG AGCTAC AGGGACC ACCAC AGGTC AGG GAACTGTCCTCCTG ATGGC AGAT CTTGTT AC AGCCCTCT CCTTGCTCTGCCTGCCCAATAT G TGTGGGCACTAGGGGTCTTGACTTGGGCTTTGCCATCCTCTACATAATACTAGTGTGAGAAG GCCCT GCCAA GTGGCTAATTTCC AGAT GGC ACCCT CACT ATAAC ACTATTAAT AGCCCC ACT GATACCCATTTCCCAAAATTTCCTGAGAGACCAGGGTGGCCTAGGGACAGCCCTTCCTGTTG AGGAGGGCTGATGGGTGTACAGGGCCCTTCCCCTGCTTCCCTGGACTTGAGCTCCAGGATC CAGCCCCAGACCAAAGATTTCCCTGTTCCCTAGGGCAACTCTGGCCCCTTTGTCTAGTTTGT a h GTAAATCTTT ATTTTTCT AGGAT ATGTTAT GCCTCCATTTCAATTAAAGTCAAGTAAAC AG ACA 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.2 Sequence for Neomycin (Source: Invitrogen plasmid) ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCG GCTATGACT GGGCACAACAGACAAT CGGCT GCTCT GATGCCGCCGTGTTCCGGCTGTCAGC GCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAG GACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTC GACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT CTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGGTGATGCAATGCGGCG GCTGC ATACGCTTG ATCCGGCT ACCT GCCC ATTCGACC ACC AAGCG AAACAfCGC ATCG AG CGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATC AGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAG GAT CTCGT CGTG AC0C ATGGCG AT GCCTGCTT GCCGAATATCATGGT GGAAAATGGCCGCT TTTCT GG ATT CAT CGACTGTGGCCGGCT GGGTGTGGCGGACCGCTATCAGGAC ATAGCGTT GGCTACCCGT GAT ATTGCT GAAG AGCTT GGCGGCGAAT GG GCTG ACCGCTTCCT CGTGCTT TACGGTAT CGCCGCTCCCG ATTCGC AGCGC AT CGCCTTCT ATCGCCTT CTTG ACGAGTT CTT CTGA 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure (3.2) show the genotyping gels for the animals selected for the electrophysiology experiments. From these genotyped mice a group of age-matched (2-4 months old) animals was chosen for experiments. Among these animals one animal each for homozygous and heterozygous mice of age 9 months were also studied to observe if aging had any affect on the muscle weakness. No differences were observed in the EMG and the muscle contractile properties of the mice in different age groups. Thus the whole set of experimental animals was pooled (such that n=18) for statistical analysis. 3.1.2 (C T G )7o o and (CTG)goo transgenic mice Lac-Z PCR amplification The (C T G )7 o o and (CTG)goo repeat mice colonies were maintained and genotyped by the lab technician and the genotyping results were provided for the animals to set up the experiments. The genotyping for Lac-Z (CTG) transgenic mice was based on the amplification of a part of the transgene. The sequence of the forward as well as the reverse primers was as follows: Forward (5’ to 3’): GCG TTA CCC AAC TTA ATC GCC Reverse (5’ to 3’): TGC ACC ATC GTC TGC TCA TCC These primers annealed to the highlighted region of the gene as shown in Table (3.3). A product of 1.15 kb was obtained by PCR amplification. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.3 Lac-Z sequence (Source: Invitrogen plasmid) AT GAT AGAT CC C GT CGTTTT ACAACGTCGTGACT GG GAAAACC CT GGCGTTACCCAACTTAA ??5§eCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGTAC CAGAAGCGGT GCCGGAA AGCTGGCTGGAGTGCGATCTTCCT GAGGCCGAT ACT GT CGT CG T CCeCT C AAACT GGCAG AT GC ACGGTTACGAT GCGCC C ATCT ACACC AACGTAACCTAT CCC ATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATT T AATGTT GAT GAAAGCT GGCT ACAGG AAGGCCAGACGCGAATTATTTTTG ATGGCGTTAACT CGGCGTTTCAT CT GTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGT CGTTT GC CGTCT GAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCT CGCGGTGAT GGT GCTGCGTTGGAGT G ACGGC AGTT ATCTGGAAG ATC AGG ATAT GT GGCGGAT GAGCGGC ATT TTCCGT GACGT CT CGTT GCTGC ATA AACCGACT ACAC AAAT CAGCG ATTTCCATGTT GCC AC TCGCTTTAAT GAT GATTT CAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGT GCGGCGAG TTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCG GCACCGCGCCTTTCGGCGGT GAAATTATCGAT GAGCGTGGTGGTTATGCCGAT CGCGTCAC ACTACGTCTG AACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCCCGAATCT CT ATCGT GCGGT GGTTGAACTGCACACCGCCG ACGGC ACGCTG ATT GAAGC AGAAGCCTGCGATGTC GGTTT CCGCGAGGTGCGGATTGAAAATGGTCT GCTGCTGCT GAACGGCAAGCCGTT GCT GA TTCGAGGCGTTAACCGT CACGAGCATCAT CCT CT GCAT GGT CAGGT CATGGATGAGCAGAC GATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATr ATCCGAACC AT CCGCT GT GGT AC ACGCT GTGCG ACCGCT ACGGCCTGTAT GT GGTGGAT GA AGCCAATATTGAAACCCACGGCAT GGTGCCAAT GAAT CGTCT GACCGATGATCCGCGCT GG CTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGT GT GAT C ATCT GGT CGCTGGG GAAT GAATC AGGCCACG GCGCTAAT C ACG ACGCGCTGTAT C GCT GG ATC AAATCT GT CG ATCCTTCCCGCCCGGT GC AGT AT GAAGGCGGCGGAGCCGAC A CCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCC GGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTG ATCCTTT GCGAAT ACGCCC ACGCGAT GGGTAACAGTCTTGGCGGTTTCGCT AAATACTGGC A GGCGTTT CGTC AG TAT CC CCGTTT ACAGGGCGGCTT C GTCTGGGACT GGGTG GAT CAGTCG CTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATAC GCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCA GCGCTGACGG AAGC AAAACACC AGC AGCAGTTTTT CC AGTTCCGTTTATCCGGGC AAACCAT CGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTG GCGCTGGATGGTAAGCCGCTGGC AAGC GGTGAAGTGCCTCTGGATGTCGCTCCACAAGGT AAAC AGTTG ATT GAACTGCCTGAACT ACCGC AGCCGG AGAGCGCCGGGCAACTCT GGCT C A C AGT ACGCGTAGT GC AACCGAACGCGACCGCAT GGTC AGAAGCCGGGCACAT CAGCGCCT GGCAGCAGTGGCGTCTGGCGGAAAACCTCAGTGTGACGCTCCCCGCCGCGTCCCACGCCA T CGCGC ATCT GACCACC AGCGAAAT GG ATTTTTGC ATC GAGCTGGGTAAT AAGCGTTGGC AA TTT AACCGCC AGT C AGGCTTT CTTTC AC AGAT GT GGATT GGCG ATAAAAAACAACTGCTGAC GCCGCTGCGCGAT CAGTT CACCCGTGCACCGCTGGAT AACGACATTGGCGT AAGT GA AGCG ACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCC GAAGC AGCGTTGTT GC AGT GC ACGGCAG ATAC ACTT GCT GATGCGGT GCT GATTACGACC G CTCACGCGT GGCAGCAT CAGGGGAAAACCTTATTT ATC AGCCGG AAAACCT ACCGG ATT GAT GGTAGTGGTCAAATGGCGATTACCGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGG CGC GGATT GGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGC GGGT AAACT GGCTCGGAT TAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCT GCC ATT GTC AGAC ATGTAT ACCCCGTACGTCTTCCCG AGCGAA 4 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.3 continued. AACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGAC TTCCAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCT GCT GCACGCGGAAGAAGGCACAT GGCT GAAT ATCGACGGTTT CCAT ATGGGGATTGGT GGC GACGACTCCT GGAGCCCGTCAGT AT CGGCGGAATTCCAGCT6AGCGCCGGT CGCT ACCATT ACCAGTT GGTCTGGTGT C AAAAAGCGGCCGCT CGAGT CTAG 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure (3.1) Six5 PCR amplification: a product of 291 bp is observed in wild type and heterozygous knock-out mice. 1) Six5{+/-) mice in lanes (1,3-5,7,8,10) 2) Six5(-/-) mice in lanes (2,11-16). 3) wild type mice in lanes (6,9,17-24) Figure (3.2) Neomycin PCR amplification: A product of 315 bp is observed in the Six5(+/-) and Six5(-/~) mice. While wild type mice showed no amplification for neomycin gene. 1) Six5(+/-) mice in lanes (1,3-5,7,8,10) 2) Six5{-/-) mice in lanes. (2,11-16). 3) wild type mice in lanes (6,9,17-24) Last four lanes show Jackson laboratory wild type mice, two Six5{-/-) founder mice and Six5{+/-) founder mice used as standard controls. Out of this set of animals 8 wild type, 7 homozygous knock-out and 3 heterozygous animals were investigated for muscle electrophysiology 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2 Data analysis 3.2.1 Six5 knock-out 3.2.1.1 Electrophysiology Electromyography Electromyogram is a test that is used to record the electrical activity of muscles. When muscles are active they produce an electric current. This current is usually proportional to the level of the muscle activity. EMG evaluates the neurophysiological function of the peripheral motor and sensory nervous system and skeletal muscles. In other words, the EMG tests if the weakness in the muscle is caused by dysfunction of the muscle or the nerves controlling the muscles. EMG is one of the most useful laboratory tests for evaluating many diseases and conditions including muscular dystrophy, inflammation of the muscles, pinched nerves, peripheral nerve damage, myasthenia gravis, disc herniation and others. There are two kinds of EMG: 1) Intramuscular EMG: involves inserting a needle electrode through the skin into the muscles whose electrical activity is to be measured. 2) And surface EMG: involves placing the electrodes on the skin overlying the muscle to detect electrical activity of the muscle. Intramuscular EMG is the classical form of EMG and was employed for the present study. Muscle tissue is normally electrically silent at rest. Once the insertion activity (caused by trauma of needle insertion) quiets down, no electrical signal is observed. Following a voluntary contraction of the muscle 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or application of external stimulus, the action potential begins to appear. As the strength of this contraction/stimulus is increased, more and more muscle fibers produce action potentials until a disorderly group of action potentials of varying rates and amplitudes (caused by complete recruitment and interference pattern) appears with full contraction/stimulus. The EMG tracings represent the sum total of electrical activity of the muscle fibers in milivolts along the Y-axis and represent time in milliseconds along the X-axis. Myotonia is defined as abnormally slow relaxation of a muscle after a contraction. Myotonic discharges were tested by subjecting the muscles of hind limb (soleus, EDL, paraspinal and intrinsic muscles of hind paw) to electromyography at room temperature under resting conditions. A total of 18 animals (age-2-4 months) were investigated that included wild type, homozygous, and heterozygous for the Six5 gene. And the total number of animals investigated for (CTG) transgenic mice, (age-14-16 months) were 23 (wild type=8, (CTG)7 0 o=7 and (CTG)9 0 o=7).The muscles were stimulated with a 30-gauge concentric needle electrode (Medtronics) as per the standard protocols (Moore et al., 1969 and Tuganowski 1972). The single microelectrode was employed to stimulate and record the stimulation of the muscle fibers as soon as 0.2-0.3 millisecs after the stimulus application. Myotonic activity was checked on insertion of the electrode needle and following the stimulation of the muscle fibers close to the area in which 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. needle electrode was inserted. There was no difference between insertional activities between animal groups. Additionally, no myotonia was seen in any muscle investigated. Muscle contractile properties The muscle fiber is composed of tightly packed subunits myofibrils that fill up most of the fiber volume. They contain the contractile elements and therefore are the structures within the muscle that responsible for force generation and active shortening. Skeletal muscle, as seen under the light microscope has a striated appearance, as seen in the picture of a myofibril (Figure 3.3). The striated appearance of is due to the arrangement of actin (thin filament; green) and myosin (thick filament; orange) and the fact that the thick and thin filaments have different refractive indices in the light microscope (the bands are called A and I). The dark bands in the (Figure 3.3) represent regions of overlap between the thin and thick filament. The region from one Z line to another is termed as sarcomere (Figure 3.4) and represents the smallest functional unit of the muscle. A sarcomere from a mammalian muscle is about 2.4 micrometer long at rest. It can be extended reversibly to more than 3 micrometers, and it can shorten to less than 2 micrometer. The appearance of the striations changes during shortening that is achieved by the actin and myosin filaments sliding over one another. According to the “Sliding filament mechanism" the cross-bridge cycling between the contractile proteins actin and myosin leads to the contraction 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure (3.3): Striated appearance of a muscle: Skeletal muscle as seen under a light microscope. The presence of A and I bands gives the muscle striated appearance. l-band H-zone M-line or bare zone Overlap region Thin filament Z-line Figure (3.4): Sarcomere :The functional unit of a skeletal muscle. Region between the two Z-lines represents one sarcomere. Thin filaments are made of actin protein and the filaments are made of myosin protein. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and relaxation of a muscle fiber. This occurs when the fiber is stimulated and the Ca2 + are released in the cytoplasm from its storage site in the sarcoplasmic reticulum. Adenosine triphosphate (ATP) provides energy for the continuous cycling of cross bridges. A twitch contraction is an experimentally induced simultaneous contraction of all of the muscle fibers contained in that muscle preparation. Therefore, all of the motor units are likewise being stimulated and firing at once in a twitch. This is true whether the stimulus is applied via the nerve of that muscle or by direct stimulation of the muscle membrane by electrode. In the later case the motor neurons may not be firing but all of the fibers associated with the motor units are being excited (depolarized) and are then contracting. A muscle twitch can be divided into different phases as shown in Figure (3.5). Figure (3.6) depicts the twitch parameters investigated in the present study. 1) Latent period: the muscle prepares itself for the contraction. Following the stimulation, the sarcolemma and the T-tubules depolarize. Ca2 + are released into the cytosol. The cross bridges begin to cycle without any visible shortening of the muscle. The duration for this phase is around 5ms. 2) Contraction phase: The myosin cross bridge cycling causes the sarcomere to shorten. This phase continues for approximately 40ms. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -T O 3 0 T»m» <m «M >e> Figure (3.5) Muscle contraction: a response to single stimulus From Sherwood, L. Human Physiology From cells to Systems. 4th ed. Brooks/Cole, CA, 2001 Tina to PaakTvwteh (TPT) Paak twitch tanskwi (Pt) ~ 2x Tim e {ms) Figure (3.6) Muscle twitch parameters: Pt represents the maximum force produced in response to single stimulus, TPT represents time taken to produce maximum twitch force, and 1 / 2 RT represents the time taken to relax such that force generated is half the Pt.. 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2) Relaxation: The Ca2 + is actively transported back into the terminal cisternae. Cross bridge cycling decreases and muscle returns to its original length thereby marking the end of a single twitch. The duration of this phase is approximately 50ms. There are three factors that affect the muscle tension developed during a twitch: 1) Frequency of stimulation 2) Number of motor units recruited 3) Degree of muscle stretch (length-muscle relationship) Present study employed the varying frequency of stimulation to study the isometric contractile properties of the muscles. A muscle may respond to a stimulus in different ways depending on the frequency of stimulation. The response of a muscle to the increasing frequency of stimulus can be explained as in Figure (3.7) and Figure (3.8) explains the tetanic parameters investigated in this study. 1) Treppe: the frequency of stimulation is so slow that there is complete relaxation between contractions. The curve goes down to the baseline after each contraction. The strength of contraction may increase because muscle contraction causes heat to build in the muscles and then muscles work better when they are warmer. This is because enzymes can work faster and more efficiently when a muscle is “warmed up”. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tem poral sum m ation C om plete tetanus Treppe Incom plete tetanus Fatigue Figure (3.7) Muscle contractions: response to increasing frequency of stimulation. 1) Treppe 2) temporal summation 3) incomplete tetanus 4) fused tetanus 5) fatigue Fatigue Index Ft = in itia l / Ffinal P P e a k tetanus tension c o % 0 ) Z 8 o u. initial Frequency (Hz) Figure (3.8) Muscle tetany parameters: Po maximum force developed in response to increased frequency of multiple stimuli. FI is denoted by the ratio of initial force developed to the final force at some point of time. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2) Temporal summation: Temporal summation or wave summation occurs when a second stimulus of the same intensity is applied to a muscle before the completion of the relaxation period of the first stimulus. This results in increased muscle tension, which may result from increased availability of intracellular Ca2 + The second peak is usually higher than the first, because the influx of Ca2 + promotes a second contraction, which is added to the first contraction. 3) Incomplete tetanus: the frequency of stimulus is increased further to a point where the muscle exhibits even shorter contraction- relaxation cycles. But there is still some degree of relaxation after each contraction. 4) Complete tetanus: When the frequency of stimulus becomes fast enough, the contractions fuse into smooth, continuous, total contraction with no apparent relaxation. This state is due to a continual deposition of the Ca2 + in the cytosol. As a result the binding sites on actin continually stay exposed. 5) Fatigue: Continued rapid stimulation of the muscle beyond complete tetanus causes muscle fatigue. Prolonged muscle activation leads to a reduction in the force producing capacity of a muscle fiber. This force reduction is attributed to reduced Ca2 + release from the sarcoplasmic reticulum, reduced Ca2 + sensitivity of myofilaments and reduced maximum Calcium-activated tension (regulated by troponin). This is linked to the reduction in intracellular pH and increase in intracellular phosphate as a 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. result of increased metabolism. In short, muscle force decreases and its response slows with fatigue. The muscle takes longer to relax after a contraction and its maximum velocity of shortening slows. Lenath-tension relationship The strength of a muscle contraction can be altered by changing the starting length the of a muscle.1) Unstretched muscle: the overlapping thin filaments from opposing ends of the sarcomere, interfere and conflict with each other. This restricts productive cross bridge building and less tension develops. The unstretched muscle produces a relatively weak contraction. 2) Moderately stretched muscle: Maximum tension is developed when there is an optimum overlap of thin and thick filaments so that all cross bridges can participate in contraction. This optimum length is termed as L0 and the study of contractile properties is carried out at this length. 3) Over stretched muscle: the thin filaments are pulled almost to the ends of the thick filaments where little tension can be developed. Stimulation of muscle under isometric (fixed length) and isotonic (fixed-load) conditions allows measurements of force production and shortening respectively. And variation in the stimulus delivered to the muscle allows comparison of twitch (single action potential) and tetanic (multiple action potential) contractions. Measurements of muscle twitches under isometric conditions allow determination of amount and time course of force development. Three variables are typically measured for the time 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. course of contraction: 1) latent period (the delay between the action potential and the start of force production) 2) contraction time (the time from start of force production to peak force) 3) and the 1/2 relaxation time (the time from peak force until the force production is 50% of the peak) For the present study we chose to study the isometric measurements investigate presence of muscle weakness. These measurements provided assessment of two basic muscle functions: 1) ability of muscles to generate force independent of size (isometric measurements normalized to unit cross-sectional area, assuming density of muscle to be 1.06 gm/cm3 ). 2) Endurance as measured by ability of muscles to sustain isometric tension for a given time period. The summarized data for muscle contractile properties are presented in Table (3.5). Both soleus and EDL muscles were investigated for the Six5 animal groups. One-way ANOVA test was used on the variables, with significances of differences between mean values assumed at p<0.05. All data are expressed as (mean ± SEM). The results can be explained as below: Optimal force-lenath relationship (U) The length of the muscle fiber was systematically measured using a micrometer while evoking single twitches in order to obtain optimal length (L0 ) for maximal twitch force production. Thereafter contractile properties were measured isometrically at L0. As given in the Table (3.5), there was 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. no significant difference between the L0 among groups of animals. Though, heterozygous soleus muscle showed slight increase in the L0, but it was found to be non-significant on statistical analysis, (p<0.05, one way ANOVA) Twitch characteristics Contraction time was measured as the time to peak twitch tension (TPT) and relaxation time was measured as the time for tension to decay from maximum to half-maximum twitch tension ( 1/2RT). For both muscles, no significant difference was found among different groups: for soleus TPT, p=0.905 and 1/2RT, p=0.772 for one-way ANOVA. Similarly for EDL, TPT p=0.955 and 1/2RT p=0.915. Peak Twitch Tension (Pt) and Peak Tetanus Tension (Pn ) Using one way ANOVA the p<0.05, no significant difference was found between different groups for Pt and P0. Peak Tetanus Tension (P0) was determined from the plateau of the force-frequency curves. P0 tended to be reduced in soleus muscle of the homozygous mice, but no significant difference was observed (p=0.111, one-way ANOVA). Except for the P0 values found in the soleus muscle of homozygous mice, all other values were within the range of published results (Dupont-Versteegden and McCarter, 1992; Lynch et al., 2001). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (3.5) Contractile abnormalities are not present in Six5 deficient mice. Soleus Lo (mm) P. (N/cm2) TPT (ms) 1/2 RT (ms) P0 (N/cm2) Fatigue Index n = WT 7.2 ± 0.3 3.7 ± 0.5 26.4 ± 2.2 96.3 ± 1 3 15.5 ± 2.2 0.61 ±0.1 8 (-/-) 7.4 ± 0.3 3.3 ±0.3 27.8 ± 2.9 90.9 ± 9.7 11.3 ± 0.9 0.67 ±0.1 7 (+/-) 7.7 ± 0 .5 - 25.7 ± 6.4 87.8 ± 12 20.3 ±6.1 0.67 ± 0.1 3 P = 0.740 0.428 0.905 0.772 0.111 0.647 Extensor digitorum longus (EDL) WT 8.7 ± 0 .3 5.0 ± 0 .5 15.1 ± 0.8 40.4 ± 4.5 20.4 ± 2.5 0.24 ±0.1 7 (-/-) 8.8 ± 0.4 6.5 ± .8 15.5 ± 1.5 39.9 ± 3.2 23.8 ±4.1 0.21 ±0.1 7 (+/-) 8.75 - 15.6 ± 1.0 42.7 ± 15 25.9 ± 0.6 0.21 ±0.1 3 P = 0.780 0.123 0.955 0.915 0.592 0.749 No significant differences were seen in optimal muscle length (L0 ), peak twitch force (Pi), time to peak twitch tension (TPT), time for tension to decay from maximum to half-maximum twitch tension (1/2RT), maximal contraction force (P0 ), or fatigue index between genotypes (one-way ANOVA). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fatigue index Muscle fatigue was induced by continuous repetitive stimulation of the muscle with 350 ms trains at 1Hz with a pulse frequency of 35 Hz and 0.2 ms duration for 10 minutes. Fatigue index was calculated by dividing the contractile force produced at 10 minutes by the initial contractile force. For fatigue index (FI), there was no significant difference among different groups of animals for both soleus (p=0.647) and EDL (p=0.749) muscles. 3.2.1.2 Histology for Six5 knock-out mice For histological studies 6 mice were selected in the two age-groups (21 /2 months and 12 months) for the Six5 knock-out mice. Histologic sections of skeletal muscle of the DM1 human patients are characterized by the presence of increased number of central nuclei, which is often termed as “Salt and Pepper” appearance. H&E sections of soleus and EDL muscles were studied to check the presence of central nuclei in the Six5 knock-out mice. Around 60 sections were observed for the presence of central nuclei, as a marker of muscle myopathy. Pictures were taken for the transverse sections using the Olympus microscope and RT slider camera (Diagnostic Instruments) at 10x and 40x magnifications. None of the sections showed centrally localized nuclei. Sections for both age group animals from all the three genotypes showed distinct nucleus on the periphery. Figure (3.9) shows one such section of the muscles each from 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (A) (B) (C) Figure (3.9) H&E sections of soleus muscle, (10x magnification). (Age-12 months). (A) Wild type), (B) Six5 homozygous knock-out, (C) Six5 heterozygous knock-out mice. No centrally located nucleus is seen in any of the sections. The arrows points to the peripherally located nucleus in each of the muscle sample. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. wild type, Six5(-/-) and Six5(+/-) mice (age group: 12 months). The results were same for both soleus and EDL muscles studied in this section. 3.2.2 (C T G )7o o and (CTG)goo transgenic mice 3.2.2.1 Electrophysiology Electromyography Myotonic discharges were tested by subjecting the muscles of hind limb (soleus and EDL) to electromyography at room temperature under resting conditions. A total of 23 animals were investigated that included wild type, (CTG)7oo and (CTG)goo transgenic mice. Myotonic activity was checked on insertion of the electrode needle and following the stimulation of the muscle fibers close to the area in which needle electrode was inserted. There was no difference between insertional activities between animal groups. However myotonia was seen in both soleus and EDL muscles, to different extents. Figure (3.10) demonstrates one of such EMG tracing depicting the EMG in both muscles investigated. Muscle contractile properties Table (3.6) gives the summarized data for contractile properties studied. Since sex of the animal made no difference to the muscle contractile properties, the data was pooled for the statistical analysis. Only soleus muscle was investigated for the (CTG)7 o o and (CTG)goo transgenic mice. The results can be explained as follows: 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Soleus } mV m i EDL 3*e rw t ilM lK T O t Figure (3.10) EMG: Demonstration of myotonia in (CTG)7o o transgenic mice: (CTG) repeat expression results in characteristic myotonia or repetitive depolarization in Soleus but milder myotonia in EDL. Calibrations correspond to horizontal (time in milliseconds) and vertical (amplitude in millivolts) intervals between two sampling points. The upper panel of tracings represent the (CTG)7 o o transgenic mice muscle EMG with long runs of myotonia, as compared to the EMG for wild-type muscle below the test EMG for each muscle. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Twitch characteristics Contraction time was measured as the time to peak twitch tension (TP T) and relaxation time was measured as the time for tension to decay from maximum to half-maximum twitch tension (1/2R T). For time to peak twitch tension (TPT) there was no significant difference among different groups, p=0.315 for one-way ANOVA. However there was a significant difference in the 1/2R T among the groups of animals. (C T G )7o o transgenic mice showed an increase of 47% over the wild type and 28% over the (CTG)goo transgenic mice. Peak Twitch Tension (Pt) and Peak Tetanus Tension (Pg) Using one way ANOVA the p<0.05, no significant difference was found between different groups for Peak Twitch Tension (Pt), (p= 0.979). Peak Tetanus Tension (P0 > ) was calculated from the force-frequency curves. There was a significant reduction in P0 in soleus muscle of the (C T G )7oo transgenic mice, (p=0.0007, one-way ANOVA). The Peak Tetanic tension (P0) was 23% lower in the (C T G )7oo transgenic mice than that of the wild type and 24.5% lower than the (CTGJgoo transgenic mice. Fatigue index Muscle fatigue was induced by continuous repetitive stimulation of the muscle with 350 ms trains at 1 Hz with a pulse frequency of 35 Hz and 0.2 ms duration for 10 minutes. Fatigue index was calculated by dividing the 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table (3.6) Contractile abnormalities in (CTG)7 oo/(C T G )c>oo mice. Soleus Pt (N/cm2) TP T (ms) 1/2 RT (ms) P0 (N/cm2 ) Fatigue Index n WT 4.38 ± 0.53 31.64±1.84 46.56 ± 4.03 23.19 ±0.91 0.50 ± .030 8 (CTGUo 4.25 ± 0.42 30.31 ±0.79 68.42 ± 6.53 17.94 ±1.05 0.65 ± .03 7 (CTG)goo 4.24 ± 0.48 28.2 ± 1.76 53.57 ± 6.42 23.9 ± 1.57 0.58 ± 0.05 7 p = 0.979 0.315 0.037 0.0007 0.031 No significant differences were seen in peak twitch force (Pt), time to peak twitch tension (TPT). But significant difference was seen in time for tension to decay from maximum to half-maximum twitch tension (1/2RT), maximal contraction force (P0), or fatigue index between genotypes (one-way ANOVA). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. contractile force produced at 10 minutes by the initial contractile force. For fatigue index (FI), there was a significant difference among different groups of animals (p=0.031, one-way ANOVA). An increase of 30% was found in the fatigue index for the (C T G )7oo transgenic mice over that of the wild type and 12% over that of the (C TG )g0o transgenic mice. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4 DISCUSSION AND CONCLUSION 4.1 Biological rationale Unlike most of the other diseases caused by trinucleotide repeat expansions, the (CTG) repeat at the DM1 locus is located in the 3’ untranslated region of the DMPK gene. This region is transcribed into mRNA but never translated into protein. Because the (CTG) repeat expansion in the DM1 locus does not alter the protein sequence encoded by DMPK, the mechanism of DM1 pathogenesis is proposed to be different from that of the polyglutamine encoding (CAG)-repeat diseases. Three different models have been proposed in order to understand this polygenic and multisystemic disease as explained in section (1.3). There is evidence for each of these mechanisms. I have tried to explore further the Chromatin structure model (mechanism 2) and RNA dominant mutation model (mechanism 3) with respect to muscle weakness. I have investigated muscle contractile properties and electromyography in transgenic mice created earlier in the laboratory, to dissect the molecular etiology of muscle weakness in DM1. 4.1.1 SIX5 and DM1 A deficiency of SIX5 mRNA in skeletal muscle from DM1 patients has been demonstrated (Klesert et al., 1997 and Thornton et al., 1997 and 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. also see Hamshere et al., 1997). Haploinsufficieny is a recognized cause of disorders resulting from mutations of the homeodomain proteins (Clapham et al., 1998 and Krapivinsky et al., 1994) and SIX5 being a transcription factor becomes important for these investigations. Deletion of Six5 leads to a variable reduction of Dmpk mRNA that may be around 50% in homozygous knock-out and 25% in heterozygous knock-out mice. It is not yet clear if this reduction in Dmpk mRNA is direct effect of loss of Six5 function or cis-effect of deletion of Six5 locus. Six5 deficient mice develop ocular cataracts, a characteristic feature of DM1 and also show reduced levels of DMPK mRNA (Klesert et al., 2000; Sarkar et al., 2000). Cardiac conduction defects that are similar to the DM1 associated cardiac conduction defects have been observed in the Six5 heterozygous mice (Wakimoto, et al., 2002). Dmpk homozygous knock-out mice develop late onset progressive myopathy (Reddy et al., 1996). The investigation of the muscle properties showed abnormal ultrastructural changes and 50% reduced force generation in these mice. I was thus intrigued to investigate similar skeletal muscle contractile properties of the mice deficient in Six5. I found no significant change in the muscle contractile function in the Six5(+/-) and Six5{-/~) mice compared to the wild type. My results thus suggest that Six5 does not play a role in the normal functioning of the skeletal muscle. Late onset progressive myopathy has been reported in Dmpk homozygous knock-out mice but not in the Six5 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. knock-out mice. This would also imply that the reduction of 25-50% Dmpk levels as observed in Six5 knock-out mice is not sufficient to result in muscle myopathy. Such that the threshold reduction of Dmpk mRNA to result in DM1 charateristic myopathy is >50% -100%. Also reported earlier, Six5 knock-out mice did not show the muscle Na+ channel gating abnormality as found in Dmpk(-/-) mice (Mistry et al., 2001). Despite all the work done towards understanding the role of Six5, it remains unclear how the deficiency of this gene leads to ocular cataract formation, cardiac conduction defects and reduced fertility. One possibility is that loss of the Six5 leads to abnormal expression of some downstream genes leading to altered ion homeostasis within some of the pathways that are involved in the normal functioning. Possibly these pathways do not affect the normal signal conduction and functioning of the skeletal muscle apparatus. Thus the reduced levels of Six5 reproduce some of the phenotype of DM1 without contributing to the DM1 associated muscle weakness. 4.1.2 (CTG) repeat overexpression and DM1 Mankondi et al. (2000) used a skeletal muscle-specific promoter to drive the expression of the transgene containing (CTG) repeat and could show the phenotype that is characteristic of DM1. Mice expressing the long repeat-transgene developed histologically defined myopathy unlike the mice that expressed short repeat-transgene. Since the only sequence similarity between the transgene and Dmpk mRNA is the (CTG) repeat, it is proposed 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that the triplet repeat is sufficient to capitulate the DM1 phenotype. In extended investigation of the same mice line, it was found that (CTG) repeat expansion triggers the aberrant splicing of the chloride channels and hyperexcitability of the skeletal muscle (Mankondi et al., 2002). The induction of aberrant splicing of the CIC-1 mRNA and the corresponding loss of the CIC-1 protein from the skeletal muscle membrane and development of hyperexcitability are all correlated with the levels of expanded (CUG) repeats in the transgenic mice. As the presence of myotonia in the transgenic mice results due to the non-functional CIC-1 channels, it has been proposed that similar deficiency of the CIC-1 conductance could be the cause of myotonia in DM1 patients. Histological abnormalities in skeletal muscle and myotonia were reported in mice transgenic for a human DMPK gene carrying an expanded (CTG)3 0 o repeat (Seznec et al., 2001). Myotonia was investigated in fore limb muscles (extensors) and hind limb muscle (soleus and EDL). These results are consistent with those obtained by Mankodi et al. (2000), that the presence of expanded (CTG) repeat is responsible for the muscle disease and myotonia. The above investigations were more focused on the histological abnormalities of the muscle and myotonia. The present study investigated muscle contractile properties and electromyography in (CTG)7 0 o and (CTG)goo transgenic mice created previously in Reddy lab. These mice 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. provided an insight into the role of (C TG ) repeat length and the dose dependence on the muscle function. In the present study, myotonia could be seen in the muscles of (CTG)7o o transgenic mice though it was more pronounced in soleus than in EDL. This is in agreement with other studies done on transgenic mice expressing (C T G ) expanded repeats. Also significant differences in the contractile properties were observed in the (C T G )7o o mice. Reduced force generation and increased 1/2RT were found to be as expected from extrapolations of the previous findings. There was an increase of 47% in the half relaxation time (1/2RT), a decrease of 23% in the peak tetanus tension (P0 ), and an increase of 30% in the Fatigue index (FI), in the (C T G )7o o transgenic mice as compared to the wild type mice. Thus there is a generalized muscle weakness in the mice expressing (C T G ) expanded repeats, with the effect being more dominant in (C T G )7o o transgenic mice. It is noteworthy that both the tetanus parameters (P0 and FI) are altered in the soleus muscle of the (CTG)7 o o mice. There could be several possible mechanisms that could lead to these changes in the muscle contractile properties. The changes observed in these mice imply that the contractile apparatus of the muscle is not able to respond to multiple stimuli in the same way as the wild type muscle. The lower P0 indicates that the muscle generates lower force in response to multiple stimuli and undergoes the fused tetanus sooner than the wild type muscle. Thus, the muscle is not 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. able to relax fast enough and stay in continued state of contraction. This is also evident from the fact that the twitch parameter 1/2RT is significantly increased in soleus of (CTG)7o o - Further, the inability of the muscle to relax normally leads to higher fatigue induction, which is evident from the significant increase in the fatigue index (FI) as compared to the wild type muscle. One of the possible reasons could be the ionic imbalance. As the muscle contraction-relaxation cycle is a process driven by depolarization and repolarization of the sarcoplasm, any perturbation of the ion channels would affect the muscle contractile properties. (CTG) repeats may play an important role in the ionic homeostasis. The (CTG) repeats have been reported to deplete crucial transcription factors by sequestration within the ribo-nucleoprotein complexes (RNP). One of such protein affected by this phenomenon is CIC-1, which could lead to myotnonia and muscle weakness. According to unpublished data, it is also observed that there is skewed distribution of muscle fiber distribution with (CTG) repeat expansion. Thus the redistribution of the fast and slow muscle fibers may alter the muscle contractile response. Lastly it may also be a possibility that the neuro-muscular junction (NMJ) is affected such that the signal conduction is altered. This can be explored by further investigation of the myotonia signals and define if the abnormality is associated with nerve conduction defect or muscle structure and function. 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The presence of milder muscle phenotype in (C T G )90o transgenic mice as compared to (CTG)roo can be explained on the basis of levels of expression at the mRNA level. The Northern blot genotyping of the transgenic mice line showed that the levels of expression of the (C T G ) repeat transgene in the (CTG)g0o mice was far low as compared to the (C T G )7o o mice. Thus the levels of expression in the (CTG)goo mice could not produce as severe muscle weakness as seen in (C T G )70o mice. This further supports that the (C T G ) repeat is sufficient to recapitulate DM1 features of muscle myopathy and there is a distinct correlation between the length and dose of the (C T G ) repeat expression. 4.2 Conclusion For the Six5 knock-out mice there was no significant difference found in the muscle contractile properties between the knock-out and the control mice. Also, no myotonia could be observed in Six5 knock-out mice. It has been reported that loss of chloride channel protein CIC-1 is sufficient to lead to myotonia in absence of other channelopathies. Since the CIC-1 channelopathy is directly associated with the (CTG) repeat expansion and the overexpression of CUG-BP, it may be possible that Six5 does not contribute to the muscle myopathy as observed in DM1. A significant decrease in the ability of the muscle to relax normally, a decrease in the force generation in response to increased frequency of stimulus, and increased fatigability, suggest that (CTG) repeat expansion 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. has a role in muscle weakness associated with DM1. Our results also show that the levels of mRNA carrying (CUG) repeat expansion and the size of this expansion is critical for the reproduction of the symptoms linked to RNA gain-of-function model. Thus these transgenic mice are useful models to help us elucidate the basic mechanism of this disease. Further investigations of the expression distribution of the (CTG) repeat containing DMPK gene and candidate genes from the DM1 locus would make it possible to draw a direct comparison between (CUG) repeat dosage, length distribution profiles and graded severity of the disease manifestation in animal models and human DM1 patients. 4.3 Future directions Most of the data available for the muscle contractile properties and DM1 phenotype are focused on the limb muscles. Differential expression of muscular dystrophy in different muscles has been reported in mice (Dupont- versteegden and McCarter 1992). Also noteworthy is that in the present study, (CTG) repeat overexpresser mice showed myotonia to different extents in the soleus and EDL muscles. Since most of the DM1 patients suffer from respiratory insufficiency and cardiac conduction defects, the evaluation of the diaphragm and the cardiac muscle functions in these transgenic mice would be of interest. The present study employed isometric stimulation of the muscles to investigate muscle contractile properties. Another approach may be to 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. study these muscles under isotonic conditions. This would enable us to assess the ability of the muscle to shorten rapidly independent of the fiber length, both as a simple value and as a rate. Measurements of shortening velocity under different loads can be used to construct a force-velocity curve. This could provide an insight into the rate of relaxation of the muscle, which would in turn help to know the status of sarcoplasmic apparatus of the muscle fibers. Presence of myotonia and differences in muscle contractile properties warrants further research of the (C TG ) transgenic mice. Detailed investigations of the muscle from these mice need to be carried to understand the myopathy. Since myotonia is observed in both the (C T G )7o o /(CTG)goo transgenic mice, nerve conduction (NC) studies should be carried out on these mice. Nerve conduction studies allows for a rapid screening for nerve conduction velocity, axon loss and defects in neuromuscular transmission. Nerve conduction studies are performed on either by stimulating brachial plexus located in the armpit of the mouse or by stimulating the sciatic nerve at the sciatic notch. Nerve conduction velocity studies (NCV) can be performed on the sciatic nerve by varying the placement of the stimulus along the length of the nerve. NCV studies give the time taken by the impulse to travel along the nerve from the sciatic notch to the ankle. Another measure, Distal Latency, gives the combination of the time taken by the impulse to conduct along the distal nerve trunk, the 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. time of neuromuscular transmission and the time of muscle fiber action potentials to sweep past the recording electrodes. These studies would allow the detection of demyelination neuropathy, axonal neuropathy, defects of neuromuscular transmission or myopathy. For further analysis of muscle functional and structural state, morphometry and histochemistry would be of prime significance. This would help in detecting the presence of other characteristic features of the DM1 myopathy, like presence of central nuclei, increase in number of nuclei, variation in muscle fiber size and reduction in myofiber density. The Dmpk knock-out mice shows adult onset progressive myopathy. The cardiac conduction defects in the Dmpk knock mice have been shown to be progressive as well. Thus, for these investigations, it is suggested to include different age groups, to determine the developmental pattern of the abnormalities. 8 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES Alwazzan M, Newman E, Hamshere MG, Brook JD: Myotonic dystrophy is associated with a reduced level of RNA from the DMWD allele adjacent to the expanded repeat. Hum Molec Genet 8:1491-1497 (1999). Amack JD, Paguio AP, Mahadevan MS: C/s and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model. Hum Molec Genet 8:1975-1984 (1999). Artero R, Prokop A, Paricio N, Begemann G, Pueyo I, Mlodzik M, Perez- Alonso M, Baylies MK : The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. Dev Biol. Mar 15;195(2):131-43 (1998). Batten FE, Gibb HP: Myotonica atrophica. Brain 32:187-205 (1909). Begemann G, Paricio N, Artero R, Kiss I, Perez-Alonso M, Mlodzik M : Muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins. Development. Nov 1; 24(21 ):4321-31 (1997). Berul Cl, Maguire CT, Aronovitz MJ, Greenwood J, Miller C, Gehrmann J, Housman D, Mendelsohn ME, Reddy S : DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model. J Clin Invest. Feb;103(4):R1-7 (1999). Berul Cl, Maguire CT, Gehrmann J, Reddy S: Progressive atrioventricular conduction block in a mouse myotonic dystrophy model. J Interv Card Electrophvsiol. Jun;4(2):351-358 (2000). Boucher CA, King SK, Carey N, Krahe R, Winchester CL, Rahman S, et al: A novel homeodomain encoding gene is associated with a large CpG island interrupted by the myotonic dystrophy unstable (CTG)n repeat. 4:1919-1925 (1995). 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Charlet-B N, Savkur RS, Singh G, Philips AV, Grice EA, Cooper TA: Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 10:45- 53 (2002). Clapham DE: The list of potential volume-sensitive chloride currents continues to swell (and shrink). J Gen Physiol 111: 623-624, (1998). Davis BM, McCurrach ME, Taneja KL, Singer RH, Housman DE: Expansion of a CUG trinucleotide repeat in the 3’ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc Natl Acad Sci, USA 94:7388-7393 (1997). Dupont-versteegden EE, and McCarter RJ: Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice. Muscle & Nerve 15:1105-1110, (1992). Ebralidze A, Wang Y, Petkova V, Ebralidse K, Junghans RP: RNA leaching of transcription factors disrupts transcription in myotonic dystrophy. Science. Jan 16;303(5656):383-7 (2004). Eriksson M, Ansved T, Edstrom L, Anvret M, Carey N: Simultaneous analysis of expression of the three myotonic dystrophy locus genes in adult skeletal muscle samples: the CTG expansion correlates inversely with DMPK and 59 expression levels, but not DMAHP levels. Hum Molec Genet 8:1053- 1060 (1999). Eriksson M, Ansved T, Edstrom L, Wells DJ, Watt DJ, Anvret M, et al: Independent regulation of the myotonic dystrophy 1 locus genes postnatally and during adult skeletal muscle regeneration. J Biol Chem 275:19964- 19969 (2000). Fardaei M, Larkin K, Brook JD, Hamshere MG : In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts. Nucleic Acids Res. Jul 1 ;29(13):2766-71 (2001). 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fardaei M, Rogers MT, Thorpe HM, Larkin K, Hamshere MG, Harper PS, et al: Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Molec Genet 11:805-814 (2002). Frisch R, Singleton KR, Moses PA, Gonzalez IL, Carango P, Marks HG, et al: Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy. Mol Genet Metab 74:281-291 (2001). Groenen P, Wieringa B: Expanding complexity in myotonic dystrophy. BioEssavs 20:901-912 (1998). Groenen PJTA, Wansink DG, Coerwinkel M, van den Broek W, Jansen G, Wieringa B: Constitutive and regulated modes of splicing produce six major myotonic dystrophy protein kinase (DMPK) isoforms with distinct properties. Hum Molec Genet 9:605-616 (2000). Hamshere MG, Newman EE, Alwazzan M, Balwinder SA, Brook JD: Transcriptional abnormality in myotonic dystrophy affects DMPK but not neighboring genes. Proc Natl Acad Sci. USA 94:7394- 7399 (1997). Harper PS: Myotonic dystrophy (WB Saunders, London, (2001). Harper, P. S. in The Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 4227-4251, McGraw-Hill Inc., New York (1995). Harris SE, Winchester CL, Johnson KJ: Functional analysis of the homeodomain protein SIX5. Nucl Acids Res 28:1871-1878 (2000). Heath SK, Came S, Hoyle C, Johnson KJ, Wells DJ: Characterisation of expression of mDMAHP, a homeodomain-encoding gene at the murine DM locus. Hum Molec Genet 6:651-657 (1997). 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IDMC: New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1). Neurology 54:1218-1221 (2000). Jansen G, Bachner D, Coerwinkel M, Wormskamp N, Hameister H, Wieringa B: Structural organization and developmental expression pattern of the mouse WD-repeat gene DMR-N9 immediately upstream of the myotonic dystrophy locus. Hum Molec Genet 4:843-852 (1995). Jansen G, Groenen PJ, Bachner D, Jap PH, Coerwinkel M, Oerlemans F, van den Broek W, Gohlsch B, Pette D, Plomp J J, Molenaar PC, Nederhoff MG, van Echteld CJ, Dekker M, Berns A, Hameister H, Wieringa B: Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nat Genet. Jul;13(3):316-24 (1996). Kawakami K, Sato S, Ozaki H, Ikeda K: Six family genes - structure and function as transcription factors and their roles in development. BioEssavs 22:616-626 (2000). Kawakami K, Ohto H, Takizawa T, Saito T: Identification and expression of six family genes in mouse retina. FEBS Lett 393:259-263 (1996). Kirby RJ, Hamilton GM, Finnegan DJ, Johnson KJ, Jarman AP: Drosophila homolog of the myotonic dystrophy-associated gene, SIX5, is required for muscle and gonad development. Curr Biol 11:1044-1049 (2001). Klesert TR, Cho DH, Clark Jl, Maylie J, Adelman J, Snider L, et al: Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nature Genet 25:105-109 (2000). Klesert TR, Otten AD, Bird TD, Tapscott SJ: Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nature Genet 16:402-406 (1997). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Korade-Mirnics Z, Tarleton J, Servidei S, Casey RR, Gennarelli M, Pegoraro E, et al: Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression. Hum Molec Genet 8:1017-1023 (1999). Krahe R, Ashizawa T, Abbruzzese C, Roeder E, Carango P, Giacanelli M, et al: Effect of myotonic dystrophy trinucleotide repeat expansion on DMPK transcription and processing. Genomics 28:1-14 (1995). Krapivinsky GB, Ackerman MJ, Gordon EA, Krapivinsky LD, and Clapham DE: Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICIn. Cell 76: 439-448 1994). Ladd AN, Charlet N, Cooper TA : The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol. Feb;21(4):1285-96 (2001). Lewis Ml, Sieck, GC, Fournier, M, and Belman MJ: Effect of nutritional deprivation on diaphragm contractility and muscle fiber size. J. Appl. Phvsiol. 60: 596-603 (1986). Liquori CL, Ricker K, Moseley ML, Jacobsen JF, Kress W, Naylor SL, et al: Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864-867 (2001). Lu X, Timchenko NA, Timchenko LT: Cardiac elavtype RNA-binding protein ETR-3) binds to RNA CUG repeats expanded in myotonic dystrophy. Hum Molec Genet 8:53-60 (1999). Lynch GS, Hinkle RT, Chamberlain, JS, Brooks, SV and Faulkner, JA: Force and power output of fast and slow skeletal muscles from mdx mice 6- 28 months old. J. Physiol. (London) 535: 591-600 (2001). Mahadevan M, Amemiya C, Jansen G, Sabourin L, Baird S, Neville C, et al: Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene. Hum Molec Genet 2:299-304 (1993). 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mankodi A, Logigian E, Callahan L, McClain C, White R, Henderson D, et al: Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289:1769-1772 (2000). Mankodi A, Urbinati CR, Yuan QP, Moxley RT, Sansone V, Krym M, et al: Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Molec Genet 10:2165- 2170 (2001). Mankodi A, Takahashi MP, Jiang H, Beck CL, Bowers WJ, Moxley RT, et al: Expanded CUG repeats trigger aberrant splicing of CIC-1 chloride channel premRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol Cell 10:35^4 (2002). Martorell L, Johnson K, Boucher CA, Baiget M: Somatic instability of the myotonic dystrophy (CTG)n repeat during human fetal development. Hum Molec Genet 6:877-880 (1997). Meola G: Myotonic dystrophies. Curr Opin Neurol 13:519-525 (2000). Michalowski S, Miller JW, Urbinati CR, Paliouras M, Swanson MS, Griffith J: Visualization of doublestranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. Nucl Acids Res 27:3534-3542 (1999). Miller JW, Urbinati CR, Teng-Umnuay P, Stenberg MG, Byrne BJ, Thornton CA, et al: Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 19:4439-4448 (2000). Mistry DJ, Moorman JR, Reddy S, Mounsey JP: Skeletal muscle Na currents in mice heterozygous for Six5 deficiency. Physiol Genomics 6:153- 158 (2001). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Monckton DG, Wong LJ, Ashizawa T, Caskey CT: Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCR analyses. Hum Molec Genet 4:1-8(1995). Moore EN, Bloom M: A method for intracellular stimulation and recording using a single microelectrode. J Appl Physiol. Nov;27(5):734-5 (1969). Murakami Y, Ohto H, Ikeda U, Shimada K, Momoi T, Kawakami K: Promoter of mDMAHP/S/x5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements. Hum Mol Genet 7:2103-2112 (1998). Napierala M and Krzyzosiak WJ: CUG Repeats Present in Myotonin Kinase RNA Form Metastable "Slippery" Hairpins J. Biol. Chem. 272:31079-31085 (1997). Neer EJ, Schmidt CJ, Nambudripad R, Smith TF: The ancient regulatory- protein family of WD-repeat proteins. Review. Nature. Sep 22;371 (6495):297-300. (1994). Ohto H, Takizawa T, Saito T, Kobayashi M, Ikeda K, Kawakami K: Tissue and developmental distribution of Six family gene products. Int J Dev Biol 42:141-148 (1998). Otten AD, Tapscott SJ: Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure. Proc Natl Acad Sci, USA 92:5465- 5469 (1995). Ranum LPW, Rasmussen PF, Benzow KA, Koob MD & Day JW: Genetic mapping of a second myotonic dystrophy locus. Nature Genet. 19, 196 (1998). Ranum LPW, Day JW: Dominantly inherited, noncoding microsatellite expansion disorders. Curr Opin Genet Dev 12:266-271 (2002). 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reddy S, Smith DBJ, Rich MM, Leferovich JM, Reilly P, Davis BM, et al: Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nature Genet 13:325-335 (1996). Sarkar PS, Appukuttan B, Han J, Ito Y, Ai C, Tsai W, et al: Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nature Genet 25:110-114 (2000). Sato S, Nakamura M, Cho DH, Tapscott SJ, Ozaki H, Kawakami K: Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1. Hum Molec Genet 11:1045- 1058 (2002). Seznec H, Agbulut O, Sergeant N, Savouret C, Ghestem A, Tabti N, Wilier JC, Ourth L, Duros C, Brisson E, Fouquet C, Butler-Browne G, Delacourte A, Junien C, Gourdon G : Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities. Hum Mol Genet. Nov 1 ;10(23):2717-26 (2001). Shaw DJ, McCurrach M, Rundle SA, Harley HG, Crow SR, Sohn R, et al: Genomic organization and transcriptional units at the myotonic dystrophy locus. Genomics 18:673-679 (1993). Schulz PE, McIntosh AD, Kasten MR, Wieringa B, Epstein HF: A role for myotonic dystrophy protein kinase in synaptic plasticity. J Neurophvsiol. Mar;89(3): 1177-86 (2003). Steinert H: Myopathologische Beitrage 1. Uber das klinische und anatomische Bild des Muskelschwunds der Myotoniker. Dtsch Z Nervenheilkd 37:58-104 (1909). Taneja KL, McCurrach M, Schalling M, Housman D, Singer RH: Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128:995-1002 (1995). 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tapscott SJ, Thornton CA: Reconstructing myotonic dystrophy. Science 293:816-817(2001). Thornton CA, Johnson K, Moxley RT: Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann Neurol 35:104-107 (1994). Thornton CA, Wymer JP, Simmons Z, McClain C, Moxley RT: Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nature Genet 16:407-409 (1997). Tian B, White RJ, Xia T, Welle S, Turner DH, Mathews MB, and Thornton CA: Expanded CUG repeat RNAs form hairpins that activate the double stranded RNA-dependent protein kinase PKR. RNA 6: 79-87 (2000). Timchenko LT, Miller JW, Timchenko NA, DeVore DR, Datar KV, Lin L, et al: Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. Nucl Acids Res 24:4407-4414 (1996). Timchenko NA, lakova P, Cai ZJ, Smith JR, and Timchenko LT: Molecular Basis for Impaired Muscle Differentiation in Myotonic Dystrophy Mol. Cell. Biol. 21: 6927-6938 (2001). Timchenko NA, Patel R, lakova P, Cai ZJ, Quan L, Timchenko LT. Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis. J Biol Chem. Mar26;279(13):13129-39. (2004). Tiscornia G, Mahadevan MS: Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios. Mol Cell 5:959-967 (2000). Tuganowski W: Simple method of stimulation and recording with a single microelectrode. J Appl Physiol. Jul;33(1):130-1 (1972). 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ueda H, Ohno S, Kobayashi T: Myotonic dystrophy and myotonic dystrophy protein kinase. Prog Histochem Cytochem 35:187-251 (2000). Wakimoto H, Maguire CT, Sherwood MC, Vargas MM, Sarkar PS, Han J, Reddy S, Berul Cl: Characterization of cardiac conduction system abnormalities in mice with targeted disruption of Six5 gene. J Interv Card Electrophvsiol. Oct;7(2): 127-35 (2002). Wang J, Pegoraro E, Menegazzo E, Gennarelli M, Hoop RC, Angelini C, et al: Myotonic dystrophy: evidence for a possible dominant-negative RNA mutation. Hum Molec Genet 4:599-606 (1995). Wang YH, Amirhaeri S, Kang S, Wells RD, GriffithJD: Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene. Science 265:669-671 (1994). Wang YH, Griffith J: Expanded CTG triplet blocks from the myotonic dystrophy gene create the strongest known natural nucleosome positioning elements. Genomics 25:570-573 (1995). Wansink DG, Wieringa B : Transgenic mouse models for myotonic dystrophy type 1 (DM1). Cytoqenet Genome Res. 100(1-4):230-42. Review (2003). Winchester CL, Ferrier RK, Sermoni A, Clark BJ, Johnson KJ: Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy. Hum Molec Genet 8:481-492 (1999). Wong LJ, Ashizawa T, Monckton DG, Caskey CT, Richards CS: Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent. Am J Hum Genet 56:114-122 (1995). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U M I N um ber: 1 4 2 4 2 2 7 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 1424227 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. 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Nautiyal, Jyoti
(author)
Core Title
Evaluation of muscle function in Six5 knock-out and (CTG) repeat overexpresser mice
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Graduate School
Degree
Master of Science
Degree Program
Biochemistry
Degree Conferral Date
2004-12
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University of Southern California
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biology, animal physiology,chemistry, biochemistry,OAI-PMH Harvest
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English
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Reddy, Sita (
committee chair
), Maxson, Robert E. (
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), Tokes, Zoltan A. (
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Nautiyal, Jyoti
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biology, animal physiology
chemistry, biochemistry