Veterinary Science - Theses
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ItemThe role of ADAM 12 in determining skeletal muscle development and intramuscular fat deposition in cattle( 2011)The research undertaken throughout this PhD thesis aimed to better understand the cellular mechanisms that influence development of skeletal muscle and intramuscular fat in beef cattle. In particular, I investigated the role of the extracellular matrix protease disintegrin and metalloprotease protein 12 (ADAM 12) in regulating muscle development and intramuscular fat deposition. These experiments sought to determine whether ADAM12 can be used as a potential modulator to manipulate these processes to increase muscling and intramuscular fat (IMF) deposition in cattle. My intital investigation sought to determine if variation in ADAM 12 expression in skeletal muscle was associated with different muscle phenotypes observed in cattle of various breeds. Primary skeletal muscle cells were isolated from Angus (high muscling), Hereford (moderate muscling) and Wagyu cross (X) Angus (low muscling) and grown in culture. The myogenic potential was found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5-20 hours in culture) in vitro. The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight of cattle. ADAM 12 gene expression was not found to be related to in vitro proliferation rates, suggesting that its influence on muscling was negligible, or that its expression in in vitro culture conditions was not reflective of its role in development of muscle tissue in vivo. The role of ADAM 12 during fat development was investigated using an in vitro model of adipogenesis, 3T3-L1 cells. ADAM 12 gene expression was knocked down over a 13-day developmental timecourse using Stealth™ RNAi. DNA, RNA and protein samples were collected to closely analyse the effect of ADAM 12 knockdown on proliferation and differentiation of 3T3-L1 cells. ADAM 12 gene knockdown was found to reduce the proliferation and differentiation of 3T3-L1 cells. However, in ADAM 12 knockdown cells enzyme markers of adipogenesis G3PDH (early adipogenic marker- glucose metabolism) and FAS (late adipogenic marker- lipid filling) were found to be greater. This was consistent with the phenotypic observation and morphometric analysis that the ADAM 12 gene knockdown treated cells displayed hypertrophy, with fewer lipids of greater size. Microarray analysis found the pathway most affected by ADAM 12 gene knockdown to be regulation of insulin-like growth factor by insulin‐like growth factor binding proteins which is thought to be responsible for the effect found on proliferation. Adipocytokines such as adiponectin and adipsin were also found to be significantly down‐regulated in ADAM 12 gene knockdown cells. Absence of these adipocytokines, in particular adiponectin, have previously been shown to cause hypertrophy of mature adipocytes due to alterations in insulin‐sensitivity and fatty acid oxidation. Thus ADAM 12 appears to be an important regulator through all stages of fat development (proliferation, differentiation and energy metabolism). Following in vitro experimentation, the role of ADAM 12 in skeletal muscle and intramuscular fat development in vivo was investigated. The m. longissimus dorsi and m. semitendinosus (LD and ST) muscles from five Angus and Brahman cattle were collected for histology and ADAM 12 gene expression analysis. Muscle fibre typing was used to determine if ADAM 12 expression patterns related to differences in skeletal muscle traits, such as muscling and IMF deposition, which are often influenced by proportions of the different muscle fibres. Consistent with other findings, the ST muscle was found to contain a higher proportion of glycolytic myofibres than the LD muscle which contained a greater proportion of oxidative myofibres. ADAM 12 gene expression was also found to be greater in the LD compared with the ST muscle. Fluorescent immunohistochemical (FIHC) staining for ADAM 12 and image analysis found that the relative amount of ADAM 12 distribution to also be greater in the LD muscle. Regression analysis found a positive relationship for the distribution of ADAM 12 against the proportion of type I myofibres (r2 = 0.86, p < 0.05) and a negative relationship for the distribution of ADAM 12 against type IIX myofibres (r2 = 0.82, p < 0.05). The gene expression and regression analysis suggest ADAM 12 is type I myofibre specific in cattle. This was further confirmed by FIHC staining of ADAM 12 with slow myosin heavy chain I (slow MHC-I) with which ADAM 12 co-localised. The type I myofibre specificity of ADAM 12 may be important in the regulation and maintenance slow myofibres in the LD and of IMF deposition, to which proportion or number of type I myofibres have been related to previously. Lastly, the expression profiles of ADAM 12 was investigated in skeletal muscle across a developmental time-course in two cattle breeds with extreme muscle phenotypes, Piedmontese X Hereford (high muscling) and Wagyu X Hereford (high marbling) cattle. Muscle biopsies (from LD muscle) were collected at 3, 12 and 25 months of age. PiedmonteseX cattle contained a greater proportion type IIX, type IIAX and type IIC myofibres compared with WagyuX cattle which had a higher proportion of the more oxidative type I and type IIA myofibres. Gene and protein expression of ADAM 12 was not different between breeds of cattle. However, ADAM 12 gene expression at 3-months of age was found to be positively related to feedlot average daily weight gain (kg/d) (r2 = 0.62, p < 0.05), liveweight at 25-months of age (kg) (r2 = 0.67, p < 0.05), hot carcass weight (kg) (r2 = 0.81, p < 0.05), retail beef yield (kg) (r2 = 0.65, p < 0.05) and eye muscle area (cm2 ) (r2 = 0.77, p < 0.05). These results suggest ADAM 12 is a candidate to be used to predict muscling and beef yield in cattle from a very young age. Analysis of ADAM 12 protein and gene expression found that expression of ADAM 12 in skeletal muscle was elevated at 3-months of age during pre-weaning muscle growth, with reduced expression at 12-months of age. Expression of ADAM 12 in skeletal muscle was upregulated again at 25-months of age, possibly related to its role in IMF deposition and marbling during this stage of growth in cattle. Thus ADAM 12 proves to be an important regulator during muscle development and IMF deposition in cattle and is a candidate to predict beef yield from a very young age. Findings from this PhD thesis show that ADAM 12 may be an important regulator of muscle and marbling development in cattle. The specific association of ADAM 12 with type I myofibres, and its role in signalling adipogenesis as outlined by this PhD thesis and work by others also suggest it is a candidate to increase marbling in cattle. Greater muscling and marbling by modulation of ADAM 12 may improve the quality and value of beef and efficiency of beef production (by increasing beef yield) to enhance competitiveness of Australia’s beef export.