Melbourne Veterinary School - Research Publications
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ItemEvidence for marsh mallow (Malva parviflora) toxicosis causing myocardial disease and myopathy in four horses(Wiley, 2017-05-01)REASON FOR PERFORMING THE STUDY: Investigation of toxicosis caused by Malva parviflora was required after 4 horses from the same farm developed severe muscle fasciculations, tachycardia, sweating and periods of recumbency leading to death or euthanasia after ingesting the plant. OBJECTIVES: To describe historical, clinical, clinicopathological and pathological findings of 4 horses with suspected M. parviflora toxicosis. The role of cyclopropene fatty acids (found in M. parviflora) and mechanism for toxicosis are proposed. STUDY DESIGN: Case series. METHODS: Historical, physical examination, clinicopathological and pathological findings are reported. Due to similarities with atypical myopathy or seasonal pasture myopathy acyl carnitine profiles were performed on sera from 2 cases and equine controls. Presence of cyclopropene fatty acids was also examined in sera of 2 cases. RESULTS: M. parviflora had been heavily grazed by the horses with little other feed available. Horse 1 deteriorated rapidly and was subjected to euthanasia. Horse 2 was referred to hospital where severe myocardial disease and generalised myopathy was determined; this horse was subjected to euthanasia 36 h after admission. Horse 3 died rapidly and Horse 4 was subjected to euthanasia at onset of clinical signs. Post-mortem examinations performed on 3 horses revealed acute, multifocal cardiac and skeletal myonecrosis. Myocyte glycogen accumulation was absent when examined in Horse 2. Acyl carnitine profiles revealed increased C14-C18 acyl carnitine concentrations in cases relative to controls. Cyclopropene fatty acids were detected in sera of cases but not controls. CONCLUSION: These findings suggest aetiology different to that of atypical myopathy or seasonal pasture myopathy. We hypothesise that cyclopropene fatty acids in M. parviflora interfere with fatty acid β-oxidation in horses in negative energy balance, causing the clinical signs and abnormal acyl carnitine profiles. These equine cases suggest a pathophysiological course that closely mimics the human genetic condition very long chain acyl CoA dehydrogenase deficiency.
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ItemImmunohistochemical analysis of laryngeal muscle of horses clinically affected with recurrent laryngeal neuropathy(WILEY, 2020-10-27)Background As myosin heavy chain (MyHC) profile of muscle fibres is heavily influenced by neural input, changes in MyHC expression are expected in horses clinically affected with recurrent laryngeal neuropathy (RLN) yet, this has not been thoroughly investigated. Objectives To describe the changes in MyHC and fibre diameter in left cricoarytenoideus dorsalis (L-CAD) muscle of horses with clinical signs of RLN. Study design Observational cohort study. Methods Immunohistochemistry was used to assess the MyHC-based fibre-type proportion, size and grouping in the L-CAD of 10 Thoroughbred horses, five clinically affected with RLN and five unaffected controls based on resting endoscopic examination. The Mann-Whitney U test was used to compare the two groups. Results Compared to controls (of mean age 3.0 ± 1.7 years) which only expressed type I, IIA and IIX MyHC, the L-CAD of affected horses (of mean age 2.8 ± 0.8 years) had obvious fibre-type grouping, and despite apparent compensatory hypertrophy of a small number of fibres, a decrease in overall fibre diameter (median difference −35.2 µm, 95% CI −47.4 to −7.9, P = .02) and diameter of type IIA fibres (median difference −46.8 µm, 95% CI −52.1 to −5.0, P = .03) was observed. Anti-fast MyHC (MY32) cross-immunoreacted with embryonic-MyHC. Whereas MY32-positive fibres were identified as type IIX in controls, in affected horses these fibres were less than 50 µm diameter with internal nuclei and were MYH3-positive for embryonic myosin indicating depletion of type IIX fibres, yet active regeneration and fibre renewal. Main limitations Small sample size that did not include subclinical cases. Fibre size and appearance rather than staining colour were relied upon to differentiate embryonic from type IIX MyHC. Conclusions Horses clinically affected with RLN have overall atrophy of fibres, loss of IIX fibres and expression of embryonic myosin indicating regenerative capacity. Despite hypertrophy of some remaining fibres, the overall decline in the bulk of fibres, including those most fatigue-resistant, may be the critical change that results in failure to maintain arytenoid abduction during exercise although direct comparison to subclinical cases is needed to confirm this.
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ItemTumour cells surviving in vivo cisplatin chemotherapy display elevated c-myc expression(NATURE PUBLISHING GROUP, 1996-03)The c-myc oncogene has been extensively implicated in cell proliferation, cell differentiation and programmed cell death. Aberrant expression of the c-myc gene product has been observed in a range of tumours and has also been implicated in cisplatin (cis-dichlorodiammineplatinum)-mediated chemoresistance. A solid transplantable tumour model in syngeneic DA rats was subjected to treatment with cisplatin to determine the impact of such therapy on endogenous c-myc gene expression. Serially transplanted tumours were intravenously treated with a single cisplatin dose (1 mg/kg) and c-myc expression analysed 2 and 7 days after treatment. The surviving tumour cells display a significant 2-fold elevation in c-myc expression at 48 h and 7 days after treatment. Primary cell cultures have been derived from untreated in vivo tumours of the same model and subjected to treatment with a c-myc phosphorothioate antisense oligomer. Administration of 5 microM c-myc antisense oligomer directed at the initiation codon and first four codons of c-myc mRNA results in total inhibition of c-myc expression and coincident suspension of cell growth for a period of 4 days in culture. Antisense therapies directed at the c-myc gene may well prove an effective tool for treating tumours in conjunction with cisplatin as these findings show that tumour cells surviving cisplatin chemotherapy display elevated c-myc expression.
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ItemCaspase-3, myogenic transcription factors and cell cycle inhibitors are regulated by leukemia inhibitory factor to mediate inhibition of myogenic differentiation(BIOMED CENTRAL LTD, 2011)BACKGROUND: Leukemia inhibitory factor (LIF) is known to inhibit myogenic differentiation as well as to inhibit apoptosis and caspase-3 activation in non-differentiating myoblasts. In addition caspase-3 activity is required for myogenic differentiation. Therefore the aim of this study was to further investigate mechanisms of the differentiation suppressing effect of LIF in particular the possibility of a caspase-3 mediated inhibition of differentiation. RESULTS: LIF dependent inhibition of differentiation appeared to involve several mechanisms. Differentiating myoblasts that were exposed to LIF displayed increased transcripts for c-fos. Transcripts for the cell cycle inhibitor p21 as well as muscle regulatory factors myoD and myogenin were decreased with LIF exposure. However, LIF did not directly induce a proliferative effect under differentiation conditions, but did prevent the proportion of myoblasts that were proliferating from decreasing as differentiation proceeded. LIF stimulation decreased the percentage of cells positive for active caspase-3 occurring during differentiation. Both the effect of LIF inhibiting caspase-3 activation and differentiation appeared dependent on mitogen activated protein kinase and extracellular signal regulated kinase kinase (MEK) signalling. The role of LIF in myogenic differentiation was further refined to demonstrate that myoblasts are unlikely to secrete LIF endogenously. CONCLUSIONS: Altogether this study provides a more comprehensive view of the role of LIF in myogenic differentiation including LIF and receptor regulation in myoblasts and myotubes, mechanisms of inhibition of differentiation and the link between caspase-3 activation, apoptosis and myogenic differentiation.
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ItemImpacts of the Callipyge Mutation on Ovine Plasma Metabolites and Muscle Fibre Type(PUBLIC LIBRARY SCIENCE, 2014-06-17)The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.
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ItemProliferation Rates of Bovine Primary Muscle Cells Relate to Liveweight and Carcase Weight in Cattle(PUBLIC LIBRARY SCIENCE, 2015-04-15)Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were 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 (P < 0.05). The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight and carcase weight of cattle (P < 0.05). Gene expression of MYF5 was also found to be significantly down-regulated in WagyuX compared with Angus cattle (P < 0.05). These findings suggest that early events during myogenesis are important for determining liveweight and caracase weights in cattle.
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ItemNutraceuticals and Their Potential to Treat Duchenne Muscular Dystrophy: Separating the Credible from the Conjecture(MDPI, 2016-11)In recent years, complementary and alternative medicine has become increasingly popular. This trend has not escaped the Duchenne Muscular Dystrophy community with one study showing that 80% of caregivers have provided their Duchenne patients with complementary and alternative medicine in conjunction with their traditional treatments. These statistics are concerning given that many supplements are taken based on purely "anecdotal" evidence. Many nutraceuticals are thought to have anti-inflammatory or anti-oxidant effects. Given that dystrophic pathology is exacerbated by inflammation and oxidative stress these nutraceuticals could have some therapeutic benefit for Duchenne Muscular Dystrophy (DMD). This review gathers and evaluates the peer-reviewed scientific studies that have used nutraceuticals in clinical or pre-clinical trials for DMD and thus separates the credible from the conjecture.
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ItemMuscle, Ageing and Temperature Influence the Changes in Texture, Cooking Loss and Shrinkage of Cooked Beef(MDPI AG, 2020-09-01)This study aimed to quantify the effect of muscle, ageing and cooking temperature on the texture, cooking loss and shrinkage of cooked beef. Cuboids from unaged (1 day post mortem) and aged (14 days post mortem) semitendinosus, biceps femoris and psoas major muscles, from both sides of five beef carcasses, were cooked at four different cooking temperatures (50, 60, 70 and 80 °C) for 30 min. and their Warner–Bratzler shear force (WBSF), cooking loss and shrinkage (longitudinal and transverse) were quantified. The WBSF was reduced by ageing in the muscles at the specific cooking temperatures: psoas major (cooked at 50, 60 and 80 °C), semitendinosus (70 and 80 °C) and biceps femoris (80 °C). The cooking loss was 3% greater in aged compared to unaged muscles. The longitudinal shrinkage was greatest in psoas major at 80 °C amongst the muscle types and it was reduced by ageing in psoas major (70 and 80 °C) and biceps femoris (80 °C). The transverse shrinkage was reduced by ageing only in biceps femoris, across all temperatures; and the diameter of homogenized fibre fragments from semitendinosus and biceps femoris was reduced more by cooking at 50 °C in unaged compared to aged condition. WBSF was related to transverse shrinkage, and cooking loss was related to longitudinal shrinkage. The effect of muscle type on the physical changes occurring during cooking of beef is dependent on ageing and cooking temperature.
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ItemKnockdown of a disintegrin A metalloprotease 12 (ADAM12) during adipogenesis reduces cell numbers, delays differentiation, and increases lipid accumulation in 3T3-L1 cells(AMER SOC CELL BIOLOGY, 2018-08-01)Mouse models have shown that a disintegrin A metalloprotease 12 (ADAM12) is implicated during adipogenesis; the molecular pathways are not well understood. Stealth RNA interference was used to knock down ADAM12 in 3T3-L1 cells. Using gene profiling and metabolic enzymatic markers, we have identified signaling pathways ADAM12 impacts upon during proliferation, differentiation, and maturation of adipocytes. ADAM12 reduced cell numbers in proliferating preadipocytes, delayed differentiation of preadipocytes to adipocytes, and increased lipid accumulation in mature adipocytes. The pathway most affected by ADAM12 knockdown was regulation of insulin-like growth factor (IGF) activity by insulin-like growth factor binding proteins (IGFBPs); ADAM12 is known to cleave IGFBP3 and IGFBP5. The IGF/mTOR signaling pathway was down-regulated, supporting a role for ADAM12 in the IGFBP/IGF/mTOR-growth pathway. PPARγ signaling was also down-regulated by ADAM12 knockdown. Gene ontology (GO) analysis revealed that the extracellular matrix was the cellular compartment most impacted. Filtering for matrisome genes, connective tissue growth factor ( Ctgf) was up-regulated. CTGF and IGBP3 can interact with PPARγ to hinder its regulation. Increased expression of these molecules could have influenced PPARγ signaling reducing differentiation and an imbalance of lipids. We believe ADAM12 regulates cell proliferation of preadipocytes through IGFBP/IGF/mTOR signaling and delays differentiation through altered PPAR signaling to cause an imbalance of lipids within mature adipocytes.
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ItemAdenylosuccinic acid therapy ameliorates murine Duchenne Muscular Dystrophy(NATURE PORTFOLIO, 2020-01-24)Arising from the ablation of the cytoskeletal protein dystrophin, Duchenne Muscular Dystrophy (DMD) is a debilitating and fatal skeletal muscle wasting disease underpinned by metabolic insufficiency. The inability to facilitate adequate energy production may impede calcium (Ca2+) buffering within, and the regenerative capacity of, dystrophic muscle. Therefore, increasing the metabogenic potential could represent an effective treatment avenue. The aim of our study was to determine the efficacy of adenylosuccinic acid (ASA), a purine nucleotide cycle metabolite, to stimulate metabolism and buffer skeletal muscle damage in the mdx mouse model of DMD. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were treated with ASA (3000 µg.mL-1) in drinking water. Following the 8-week treatment period, metabolism, mitochondrial density, viability and superoxide (O2-) production, as well as skeletal muscle histopathology, were assessed. ASA treatment significantly improved the histopathological features of murine DMD by reducing damage area, the number of centronucleated fibres, lipid accumulation, connective tissue infiltration and Ca2+ content of mdx tibialis anterior. These effects were independent of upregulated utrophin expression in the tibialis anterior. ASA treatment also increased mitochondrial viability in mdx flexor digitorum brevis fibres and concomitantly reduced O2- production, an effect that was also observed in cultured immortalised human DMD myoblasts. Our data indicates that ASA has a protective effect on mdx skeletal muscles.