Increased calcium-activated calpain proteolysis in the sarcolemma membrane is thought to be a primary mechanism in the pathophysiology of Duchenne Muscular Dystrophy (DMD). However, few studies have tested this possibility prior to the overt signs of the dystrophy. The purpose of this study was to test the hypothesis that there is greater calpain content and total relative calpain activity in membranes obtained from dystrophic (mdx; mdx:utrophin-deficient (mdx:utrn-/-)) compared to wildtype (wt) mouse skeletal muscles during maturation at ages 7- and 21-d,and at a post-maturation age of 35-d. Calpain activity was determined as the calcium-dependent cleavage of the flurogenic substrate SLY-AMC, and content was determined by Western analysis with an anti-calpain antibody. There were several intriguing findings: 1. There was an inverse relationship between calpain content and relative activity in the whole muscle in both wt and mdx mice from age 7- to 35-d: calpain content decreased, and relative calpain activity increased as the mice aged. This suggests a similar role for calpain in both genotypes, which might relate to specific maturation processes, possibly up to age 21-d. Although the inverse relation was evident at 35-d, the targets for calpain in mdx compared to wt likely differed. 2. The increased relative calpain activity in the membrane fraction of mdx mice at age 35-d (26.73 Arbitrary Units, (AU)) compared to that of age 7- (4.9AU; p<0.05) and 21-d (8.74AU; p<0.05) is temporally related to degeneration and regeneration processes, and may also indicate activation of apoptosis, in mdx muscles at this age. 3. At age 7-d, there were no significant differences in either calpain content or relative calpain activity in all subcellular fractions for wt and mdx mice. This result might suggest similar calpain distribution and activities that are related to the regulation of muscle maturation and differentiation in both genotypes. (Note:data were not obtained for the mdx:utrn-/- mice at age 7-d because of insufficient animals). 4. At age 21-d, there was greater relative calpain activity in the myofibrillar supernatant fraction in mdx (15.13AU) than wt mice (1.18AU; p<0.05). This could indicate calpain's role in the initiation of myofibrillar protein turnover and the proteolysis of submembranous networks in the mdx muscles. 5. At age 21-d, greater calpain content in the mdx (1.40ìg) compared to wt (0.23 ìg; p<0.05) membrane fraction might suggest a broader distribution of calpain along membranes that contributes to the onset of dystrophy in the mdx muscles. 6. At age 35-d, there was greater calpain content in the mdx:utrn-/- compared to the wt membrane (0.48ìg vs 0.13 ìg), cytosolic (0.88ìg vs 0.30ìg), and myofibrillar supernatant (0.49ìg vs 0.17ìg; p<0.05 ) fractions This increased content and broad distribution across several subcellular fractions may reflect degeneration and regeneration processes, and potentially activation of apoptosis, in the mdx:utrn-/- muscles. These data suggest that calpain activity contributes to dystrophic pathophysiology mainly in the membrane fraction of mdx skeletal muscles at age ~21-d, but appears to contribute later at 35-d and in more subcellular fractions in mdx:utrn-/- skeletal muscles. / Master of Science

MSc (Med), Genetic Counselling, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are severe X-linked recessive, degenerative neuromuscular diseases. DMD/BMD are caused by deletions, duplications and point mutations in the DMD gene situated on the X-chromosome. Studies have shown that the risk of being a carrier for DMD/BMD has a psychosocial impact on individuals and affects their requests for DNA testing and their choices regarding reproduction. Very few articles have been published to date and this study is the first South African study to investigate the behaviours of individuals in DMD/BMD families. The study aimed to investigate why individuals attended genetic counselling and who referred them. It also aimed to identify factors that influence at-risk individuals‟ decisions regarding genetic counselling, carrier testing and reproduction. The study was retrospective and data were obtained by reviewing genetic counselling files at the Division of Human Genetics, National Health Laboratory Service and the University of the Witwatersrand. The sample consisted of 79 files of families seen for genetic counselling regarding DMD/BMD from 1995 to 2008. Subjects included the maternal female relatives of affected individuals, who were all of reproductive age (15-49 years); the total number of at-risk individuals identified was 237. Subjects were divided into three groups according to their assigned reproductive risks: low (0-9%), intermediate (10-24%) and high (>25%). The influence of reproductive risk and other identified variables on decisions to attend genetic counselling, have carrier testing and having children were analysed using chi-squared and logistic regression analysis. iii Reproductive risk and relationship to the affected individuals were shown to be significant predictors of individuals‟ decisions. Other factors that contributed significantly to the behaviour of at-risk individuals were ethnicity, age, whether a mutation was de novo and whether an individual had affected children.

AIMS: To evaluate the use of Manual Muscle Strength Tests (MMST), Timed Functional Tests (TFT) and Respiratory Function Tests (RFT) as measures of muscle strength in young boys with Duchenne Muscular Dystrophy (DMD) and specifically to evaluate the use of Peak Expiratory Flow (PEF). BACKGROUND: There is a need to measure the effect of treatments that potentially increase muscle strength in DMD. PEF may have advantages over Vital Capacity (VC) as a measure of respiratory function in young boys with DMD. METHODS: 17 boys with DMD (aged 5-10 years) were assessed regularly over one year. Assessment involved Respiratory Function Testing (PEF, VC, Forced Expiratory Volume in one second [FEV1]), Timed Functional Testing (walking 9 metres, climbing four stairs, arising from supine) and MMST. A single investigator performed MMST and TFTs. A separate investigator performed RFTs. For RFTs a percentage of predicted was calculated [PEF(%), FEV1(%), VC(%)].11/17 boys were treated with prednisolone which increases strength in DMD. RESULTS: At baseline, all boys had significant weakness. Mean (+/- SD) PEF(%) 69 +/- 13% and VC(%) 77 +/- 18% were abnormal. Baseline PEF(%) predicted correlated with MMST (P=0.003) and time to walk 9 metres (P=0.022). Baseline VC(%) correlated with MMST (P=0.049). There was a consistent statistically significant correlation between MMST and all TFTs. PEF was performed well on 80% of occasions, spirometry on 65%. Changes in PEF(%) showed statistically significant correlation with changes in all TFTs. The correlation was not statistically significant for VC(%) or FEV1(%). Prednisolone treated boys did better than those not treated. PEF, time to walk 9 metres and time to climb 4 stairs showed statistically significant improvement. The mean improvement from baseline in PEF(%) was 19 +/-14% in treated and 2 +/- 7% in untreated boys (P=0.012). CONCLUSIONS: MMST, TFTs and RFTs are valid measures of muscle strength in young boys with DMD. PEF is abnormal in young boys with DMD; correlates with other measures of strength and is sensitive to changes in strength. PEF is more easily performed than spirometry and has a role in monitoring muscle strength in young boys with DMD.

Oculopharyngeal Muscular Dystrophy (OPMD) is a late-onset dominant/recessive myopathy caused by the expansion of a polyalanine repeat in exon 1 of the PABPN1 gene. The expression of expanded PABPN1 (expPABPN1) triggers the formation of insoluble nuclear aggregates within muscle fiber nuclei of OPMD patients. These aggregates are enriched in poly(A)RNA and sequester molecular chaperones, ubiquitin and proteasome subunits. In addition to these cellular components, we first identified two novel PABPN1 interacting partners, hnRNPA1 and hnRNPA/B that also localized to the insoluble expPABPN1 aggregates. However, only hnRNPA1 was observed in inclusions of OPMD patients' muscle fiber nuclei. Following this finding, we next established the involvement of the ubiquitin-proteasome pathway in the clearance of misfolded expPABPN1 and provided more insights into the beneficial role of molecular chaperones in OPMD. The inhibition of proteasome correlated with an increase in the aggregation of expPABPN1, suggesting a possible proteasome impairment in OPMD. Conversely, the overexpression of Hsp70 and Hsp40 coincided with a decrease in nuclear aggregates concomitant with a reduced cellular toxicity, suggesting the therapeutic potential of manipulating molecular chaperones levels. Finally, we demonstrated that soluble forms of expPABPN1 are the primary toxic species in OPMD. In the presence of endogenous HSPs, a decrease in expPABPN1 aggregation correlated with an increased cellular toxicity. A defect in polyadenylation or ubiquitination significantly increased expPABPN1 solubility and cell death. Using live-cell imaging, we observed that nuclear aggregates prolonged the survival of expPABPN1-expressing cells, which led us to speculate that protein aggregates are subnuclear structures that preserve cellular homeostasis by depleting the expPABPN1 from the nuclear soluble pool. We propose that the polyalanine expansion in expPABPN1 could enable aberrant protein-protein interactions that would compromise the cellular function of nuclear factors and the expression of genes essential for muscle integrity and differentiation. For instance, expPABPN1 might compromise the function of hnRNP proteins and lead to altered mRNA processing and nucleocytoplasmic export, which can be detrimental to the cell.

Thesis (M. Sc.)--University of New South Wales, 2003. / Also available online.

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