A traumatic skeletal muscle injury that involves the loss of a substantial portion of tissue will not regenerate on its own. Little is understood about the ability of the muscle to recover function after such a defect injury, and few research models exist to further elucidate the repair and regeneration processes of defected skeletal muscle. In the current research, a model of muscle injury was developed in the lateral gastrocnemius (LGAS) of the rat. In this model, the muscle gradually remodels but functional recovery does not occur over 42 days. Repair of the defect with muscle-derived extracellular matrix (ECM), improves the morphology of the LGAS. Blood vessels and myofibers grow into the ECM implant in vivo, but functional recovery does not occur. Addition of bone marrow-derived mesenchymal stem cells (MSCs) to the implanted ECM in the LGAS increases the number of blood vessels and regenerating myofibers within the ECM. Following 42 days of recovery, the cell-seeded ECM implanted LGAS produces significantly higher isometric force than the non-repaired and non-cell seeded ECM muscles. These results suggest that the LGAS muscle defect is a suitable model for the study of traumatic skeletal muscle injury with tissue loss. Additionally, MSCs seeded on an implanted ECM lead to functional restoration of the defected LGAS. / text

The intrinsic laryngeal muscles are recognized as a highly specialized allotype of skeletal muscle. To date, much of the research examining the properties of this muscle group has been conducted on 2 primary muscles: the thyroarytenoid and posterior cricoarytenoid. Consequently, it is unknown whether the remaining intrinsic laryngeal muscles evidence this highly refined phenotype or if they retain a phenotype more similar to prototypical skeletal muscle. The purpose of this study was to further define the biologic properties of the interarytenoid (IA) and cricothyroid (CT) muscles of the larynx using the dystrophin deficient mdx mouse model. Previous work in this model has demonstrated sparing of select craniofacial muscles in the disease. Interestingly, a vast body of literature also supports the uniqueness of these spared muscles in a number of other areas including: fiber types, motor unit size, proprioceptive mechanisms, myosin isoform expression, remodeling behaviors, and sarcomeric structure. It follows, then, that muscle response to dystrophin deficiency serves as a sensitive marker of a muscles level of biological specialization and its similarity to or departure from classic limb muscle. Larynges and gastrocnemius muscles from 8 mdx and 8 C57BL control mice were examined histologically for typical markers of dystrophinopathy. Immunocytochemical testing examined the distribution of dystrophin and its homolog, utrophin, in control and mdx muscles. Results demonstrated that despite the absence of dystrophin, the laryngeal muscles did not show the classic markers of disease. The mdx superior cricoarytenoid muscle (SCA; mouse counterpart of human IA) demonstrated no evidence of damage, inflammation, necrosis, or regeneration. The mdx CT evidenced subtle markers of regeneration (eg, slight increase in centrally nucleated fibers) but no evidence of degeneration. The authors concluded that the SCA was spared from the effects of dystrophin deficiency, while the CT was strongly protected. The results demonstrate that the SCA and CT muscles of the larynx possess a specialized nature that separates them from prototypical limb muscle. Information from the study offers insight into the unique biology of the laryngeal muscles and holds implications for the translational study of voice and voice disorders.

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The aims of this thesis were to 1) investigate the effect of SB431542 <i>in vitro</i> and <i>ex vivo</i> as a novel approach towards promoting the functional hypertrophy of skeletal muscle by inhibiting the myostatin-Smad pathway, 2) to investigate the expression and function of the Yes-associated protein (Yap) in skeletal muscle and C2C12 cells as a novel regulator of C2C12 differentiation and 3) to generate a GFP-RCASBP-hYAP1 S127A retrovirus to allow the study of the function of Yap in skeletal muscle differentiation <i>in vivo</i>. The results presented in this thesis show that SB431542 promotes the hypertrophy of C2C12 myotubes and mature <i>Xenopus</i> skeletal muscle fibres. However, SB431542 treatment also results in a reduction in specific force of mature <i>Xenopus</i> muscle fibres suggesting that SB431542 is not suitable as a treatment for skeletal muscle atrophy. These results also show that Yap is expressed in mouse skeletal muscle <i>in vivo</i> and that Yap is a novel regulator of C2C12 differentiation. Finally, these results descried the generation of a GFP-RCASBP-hYAP1 S127A retrovirus that can be used to assess the role of Yap <i>in vivo </i>during skeletal muscle formation in the chick embryo. Together, these results suggest that Yap is a novel regulator of C2C12 differentiation that should be studied as a potential therapeutic target in musculoskeletal diseases.

Background: Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity. Results: Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 +/- 0.7 kg/m(2)) and obese (n = 10; BMI = 32.9 +/- 0.7 kg/m(2)) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change -1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5' UTR (Chr. 8: 22,423,530-22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity. Conclusions: These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.

OBJECTIVE: To compare skeletal maturation of female and male subjects from historic samples to present day subjects by assessing Fishman’s Skeletal Maturity Index (SMI). Present day eating habits and lifestyle have been suggested as factors in accelerating pubertal maturation seen within the last century. Consequently, Body Mass Index (BMI) as well as BMI percentile were evaluated to determine whether it is associated with significant differences in skeletal maturation patterns. METHODS: This pilot study included hand-wrist films from 92 subjects from the Burlington and Forsyth longitudinal growth studies (1959-1970) and 146 patients currently enrolled in the Orthodontic department of the Boston University Henry M. Goldman School of Dental Medicine (BUGSDM). The age of the subjects ranged between 7 and 16 years. SMI stage was determined and BMI and BMI percentile were calculated for each subject. RESULTS: The mean chronologic ages of all the SMI stages were not different in males or females when comparing the historic sample to the present sample except for the mean ages at SMI stage 7 and 11 for females and SMI stage 5 for males. Females in the present sample reached SMI stages 7 and 11 significantly earlier: 11.6 versus 13.3 years for SMI 7 (p<0.001) and 15.6 vs. 16.0 for SMI 11 (p<0.05). Males in the present sample also reached SMI 5 significantly earlier: 12.5 vs 13.8 years (p<0.05). It was also seen that present day females at SMI stage 11 not only matured earlier, but also had significantly higher mean BMI and mean BMI percentile than the females at the same stage in the historic sample. This suggests that BMI may be associated with acceleration of maturation among females at SMI stage 11 (p<0.05). CONCLUSIONS: In both male and female subjects, there are differences between past and present populations at certain SMI stages. This evidence suggests that patterns of skeletal maturation may have changed and BMI may be associated with such changes. The differences in skeletal maturation between the two groups may also be a result of the different distribution of race in each group. In the current study, the historic sample consists of only Caucasian subjects whereas the present sample consists of subjects from many different backgrounds. As a result, effects of racial variations could have additionally played a role in the changes seen in skeletal maturation patterns. Increasing our sample size and controlling for race may help further elucidate these changes and determine if this transition towards earlier maturation is in fact due to increasing BMIs.

To prevent damaging ATP depletion during periods of intense activity‚ intrinsic mechanisms within skeletal muscle are activated and lead to myoprotection; a process known as muscle fatigue. It has been proposed that the primary mechanism of fatigue is a submaximal sarcoplasmic reticulum Ca2+ release and decreased force generation‚ however‚ what triggers this mechanism remains controversial. It is possible that glycogen may act as a trigger as studies have repeatedly shown a direct correlation between glycogen content at the beginning of activity and time to fatigue. In previous studies‚ a fatigue bout and/or period of fasting to deplete glycogen was used. However, this leaves investigators to differentiate between the effects of glycogen depletion methodology causing a metabolic stress and effects of glycogen itself. One objective of this M.Sc. project was to produce a low glycogen model without a prior metabolic stress that could forgo these limitations. It was extended to differentiate between the role of glycogen and extracellular glucose during fatigue as well as fatigue pre-conditioning (FPC)‚ a phenomenon in which fatigue resistance increases for about 2 hours after a first fatigue bout. During a single‚ first fatigue bout (one contraction every sec for 3 min) a mean decrease in glycogen from 95 to 20 μmol/g dry wt. had no effect on the decrease in tetanic [Ca2+]i‚ i.e. the [Ca2+]i during tetanic contractions‚ whereas removing glucose from the physiological solution led to a 46% greater decrease in tetanic [Ca2+]i than when glucose was present. During a subsequent fatigue bout (i.e. FPC) a greater amount of glycogen was used as glycogen content was 27% greater than prior to the first fatigue bout. When glycogen and/or glucose was limited‚ FPC was abolished. It is concluded that extracellular glucose is critical to prevent fatigue. Additionally, whereas glycogen is important for FPC‚ it appears to be much less important during a first fatigue bout initiated in absence of any prior metabolic stress.