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Analysis of Satellite Cells in Human and Rat MusclesIdris Sidique1, Dr R Billeter-Clarke1, Prof P L Greenhaff1 School of Biomedical Sciences, University of Nottingham, Nottingham, NG7 2UH Background: Satellite cells are muscle specific stem cells that have been the point of much discussion since their discovery by diMauro in 1961. Found beneath the basement membrane of adult skeletal muscle fibres, they are involved in the process of skeletal muscle regeneration. The regeneration response, often as a result of mild exercise and or myotrauma is preceded by satellite cell activation, proliferation and differentiation into skeletal muscle myoblasts, which either directly fuse together to replace injured muscle fibres or fuse to existing muscle fibres, hence contributing myonuclei during muscle turnover. As such, an in-depth understanding of the intricacies of the regeneration response, especially those factors that influence on satellite cell activation has significant clinical application. Methods: Satellite cells are between 6 and 8m long and based on the observation that these cells are often cut in half in muscle cryostat cross-sections, identifying those cells that are in the cutting plane of two adjacent sections provides a means of running multiple analysis on the same cell whilst preserving the integrity of the cell’s genetic content. Muscle cross-sections (8-10m) were cut and flipped to expose the same cutting surface between two consecutive sections; allowing one section to be used for an independent experiment (e.g. Laser Capture Microdissection, in-situ hybridisation (ISH), etc) and the other with immunocytochemistry (for pax7 or CD56) to identify satellite cells. Using light or fluorescence microscopy, satellite cells on cross-sections were visualised and matched to a partner on the adjacent section. Results and Conclusions: In the rat muscles, 23.5±4.7% of pax7+ satellite cells could be matched on both sections. Pax7 is a nuclear antigen, staining with a membrane antigen (CD56/NCAM, a cell-cell recognition glycoprotein) yielded a higher percentage of matched cells on both sections (36.7%±0.14); the highest possible numbers of satellite cells in the cutting plane that could be matched to adjacent muscle sections. This is not ideal for investigating transcripts that are suspected to be expressed at a higher level in muscle fibres that satellite cells, equally the method is sufficient for transcripts that are alleged to be highly expressed in satellite cells than muscle fibres and one of these is myostatin; a negative regulator of satellite cells. We are in the process of setting up ISH for myostatin in order to investigate its expression in normal an atrophying/regenerating muscle. MOLECULAR MECHANISMS UNDERLYING THE EFFECT OF PIOGLITAZONE THERAPY IN NON-ALCOHOLIC STEATOHEPATITIS (NASH) Abdul Rahim R1, Aithal GP2, Macdonald IA1, Bennett AJ1,2. 1School of Biomedical Sciences, University of Nottingham Medical School, 2Nottingham Digestive Diseases Centre: Biomedical Research Unit, Nottingham, UK
Methods: Liver biopsy samples collected from 46 non-diabetic subjects before and after 1-year treatment with 30mg/day of Pioglitazone or placebo were used for RNA and protein extraction. 200ng of total RNA was used to synthesise cDNA for gene expression using Taqman Real-Time Polymerase Chain Reaction (RT-PCR). Results:
Table 1: Effects of 12 months of Pioglitazone treatment on gene expression. Gene expression values are normalised using the geometric mean of the reference genes (beta actin and hydroxymethylbilane synthase). Data are presented as mean ± SEM. Carbohydrate regulatory element binding protein (ChREBP), Sterol regulatory element binding protein-1C (SREBP-1C), Carnitine palmitoyl transferase-1 (CPT-1), Pyruvate dehydrogenase kinase 4 (PDK4), Pyruvate kinase 2 (PK2) and Glucokinase (GCK) (a: Pre-trial Vs Placebo; b: Pre-trial Vs Pioglitazone; c: Pioglitazone Vs Placebo) Conclusions: Pioglitazone therapy caused a significant upregulation of both ChREBP and SREBP-1C gene which are both involved in the transcriptional regulation of lipogenesis. Conversely, patients given placebo showed marked reduction in the SREBP-1C by the end of the trial period. It is unclear at present as to the mechanism by which Pioglitazone therapy increases lipogenic transcription factor levels. These findings may reflect the improved whole body insulin sensitivity observed in the Pioglitazone treated group. Alternatively the increase in ChREBP and SREBP-1C may be caused by direct effects of Pioglitazone activation of PPAR-γ (Peroxisome proliferator-activated receptor- γ) in the liver. Depletion of carnitine in rat myocardium and skeletal muscle by oral mildronate administration C. Porter, D. Constantin-Teodosiu, S.M Gardiner and P.L. Greenhaff. School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, UK Background and aims: Mildronate is a structural analogue of the carnitine precursor, gamma-butyrobetaine, that inhibits the activity of gamma-butyrobetain-hydroxylase, thereby decreasing carnitine biosynthesis1 and presumably mitochondrial long-chain fatty acid transport. The attenuation in muscle long chain fatty acid oxidation in insulin resistance and intense exercise has been associated with the decline in muscle free carnitine availability observed under these conditions2,3, but evidence is lacking showing an unequivocal causative link. The aim of this study therefore was to develop a robust in vivo animal model that would permit the investigation of the impact of tissue carnitine depletion on energy metabolism and function. Materials and methods: Sixteen male Hans Wistar rats (mean body mass 310 g) were divided into 2 groups that received either drinking water (Control, n=8) or drinking water supplemented with mildronate (Mildronate, n=8) for 10 days (1,600 mg/day/kg on days 1-2 and 800 mg/day/kg thereafter). After 10 days, the myocardium, extensor digitorum longus (EDL), gastrocnemius (GAS), soleus (SOL) and tibialis anterior (TA) muscles (chosen for their differences in fibre composition), were removed under terminal anaesthesia (sodium pentobarbital, i.p.), freeze-clamped immediately and stored at -80°C. Muscle free, long and short chain acyl-carnitine content were determined using radioenzymatic methods and total carnitine was calculated as the sum of these sub-fractions. All animal procedures were approved by the UK Home Office and carried out in accordance with the Animals (Scientific Procedures) Act (1986). Results: Mildronate administration resulted in a marked reduction in carnitine-sub-fractions in all tissues, but the reduction in free carnitine content was particularly remarkable (% -90, -88, -86, -84 and -88 in the heart, EDL, GAS, SOL, TA respectively; Table 1). Conclusions: The results demonstrate that the mildronate dosing regimen used in the present study provides an ideal model to investigate the significance of carnitine availability on metabolic regulation and physiological function in myocardium and skeletal muscle in vivo. Table 1: Tissue carnitine sub-fractions and total carnitine content in control and mildronate treated Hans Wistar rats.
Values expressed as means ± SEM (n=8). Concentrations are expressed as mmol/kg of dry muscle. *, *** Significantly different from the corresponding control group (P<0.05; P<0.001, respectively, student’s t-test). References: 1. Simkhovich et al. (1988). Biochem pharmacol 37, 195-202 2. van Loon et al. (2001). J Physiol 536, 295–304. 3. Blaak. (2004). Proc Nutr Soc 63, 323–330. This work was funded by a BBSRC Case Award with AstraZeneca Pharmaceuticals Studies of the atrogene ZNF216 in skeletal muscle atrophy Joanna Strachan, James R. Cavey, Sheila M. Gardiner, Paul L. Greenhaff and Robert Layfield Skeletal muscle atrophy is a common feature of a number of clinical conditions including sepsis, cancer and AIDS. Protein is lost from atrophying muscle in part via increased activity of the ubiquitin-proteasome system. Genes that are up-regulated in atrophying muscle and directly mediate the atrophy programme are termed ‘atrogenes’ and the E3 ubiquitin ligases MAFbx and MuRF1 have been characterised as atrogenes in various atrophy models. Whilst upstream pathways that regulate MAFbx and MuRF1 expression are relatively well characterised, pathways downstream of these E3s are yet to be fully elucidated. ZNF216 has more recently been characterised an atrogene and its expression has been shown to be up-regulated in denervation and fasting-induced models of muscle atrophy. The ZNF216 protein contains A20 and AN1 domains at its N- and C-termini, respectively, the former having the ability to bind polyubiquitin chains. ZNF216’s reported association with the 26S proteasome suggest it may function downstream of MAFbx and MuRF1, potentially shuttling proteins targeted (by these E3s) for degradation. This study aims to characterise ZNF216 expression (mRNA and protein) in an LPS-infused rat model of sepsis and to identify binding partners (potential substrates) of ZNF216 in skeletal muscle. Chronically-instrumented male Sprague-Dawley rats were continuously infused intravenously with either LPS (15 μg kg-1 h-1; n=8), dexamethasone (12.5 μg kg-1 h-1; n=8) which was administered simultaneously with LPS or saline (n=8) for 24 h at 0.4ml h-1. The extensor digitorum longus (EDL) and gastrocnemius muscle was then removed and freeze-clamped under terminal anaesthesia. Procedures were approved by the University of Nottingham Ethical Review Committee and were performed under Home Office Project license authority. Comparisons were made between LPS and control mRNA levels (obtained using RT-PCR.) Recombinant wild type GST-rZNF216 (rat sequence) and A20 domain mutant (C30/33A) were purified using glutathione-Sepharose and immobilised on beads to compare their affinity for K48 and K63 polyubiquitin chains. ZNF216 binding proteins from rat gastrocnemius muscle were identified using immobilised wild type GST-rZNF216 in pull-down assays. ZNF216 was upregulated >2 fold, MAFbx ~4 fold and MuRF1 >10 fold in LPS infused EDL in comparison with control, further substantiating the notion that ZNF216 is a bona fide atrogene. Increases in ZNF216 expression were not blunted by a low dose of dexamethazone, a treatment known to spare muscle atrophy in this model. Immobilised wild type GST-rZNF216 bound polyubiquitin chains and mutation of the A20 domain (C30/33A) abolished ubiquitin-binding. Polyubiquitinated proteins from gastrocnemius bound selectively to wild type ZNF216; direct identification of these ubiquitinated muscle proteins was attempted following further fractionation by reverse phase-solid phase extraction (RP-SPE) and 2DE. Future studies will attempt the identification of ubiquitinated binding partners of ZNF216 via selective elution with deubiquitinating enzymes, and ultimately will aim to catalogue the complement of ubiquitinated proteins in resting and atrophying skeletal muscle in order to identify substrates of the ‘atrophy programme’. Effects of sustained inhibition of FAAH on endocannabinoid synthesis and catabolism in the midbrain and liver Bright Okine Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase enzyme which hydrolyses fatty acid ethanolamides (FAEs) including anandamide (AEA) and N-palmitoylethanolamine (PEA), produced primarily by the action of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD). Acute inhibition of FAAH by the irreversible inhibitor URB597 increases levels of AEA and produces an analgesic phenotype (Jhaveri et. al., 2008). The impact of sustained inhibition of FAAH on endocannabinoid turnover is unknown. Herein, the effects of URB597 on levels of FAAH, NAPE PLD (FAE synthetic enzyme), and MAGL (monoacylglycerol lipase, which hydrolyses the endocannabinoid 2-arachidonoylglycerol, 2-AG) mRNA and protein expression, FAAH enzyme activity and levels of AEA, PEA and 2-AG are reported. Adult male Sprague-Dawley rats (200-250g) received intraperitonial injections of URB597 (0.3 mg kg-1) or vehicle on four consecutive days. Rats were killed on day 4 and midbrain and liver samples were collected and snap frozen in liquid nitrogen. FAAH, NAPE-PLD and MAGL mRNA in each tissue was determined, relative to β-actin, using Taqman-based real-time PCR, while protein levels were assayed using immunoblotting. FAAH activity (measured as the hydrolysis of 100 μM oleamide) and tissue levels of AEA, PEA and 2AG were measured using a modified LC-MS-MS method based on previous work (Richardson et al, 2007). Statistical analysis on data from 5-6 rats was conducted using 1-way ANOVA with Bonferonni’s multiple comparison test (for FAAH activity data) unpaired T-test (for mRNA and protein data) or Mann-Whitney U test (for endocannabinoid data). 4 day dosing with URB597 significantly elevated levels of PEA in the midbrain (from 0.22±0.053 to 0.89±0.038 nmol/g) and liver (0.10±0.037 to 0.28±0.013 nmol/g) (P<0.01 for both). Levels of AEA in the midbrain were elevated (55±35 to 126±62 pmol/g; P=0.05) but those in liver were unchanged. Levels of 2-AG were unaltered following URB597 treatment in either the midbrain or liver. URB597 treatment significantly decreased FAAH activity in the midbrain (0.16 ± 0.06 nmol min-1 mg-1 protein, P<0.05) and liver (0.29 ± 0.04, P<0.001) compared to vehicle-treated rats (0.97 ± 0.32 and 1.20 ± 0.13, respectively). URB597 treatment significantly reduced FAAH mRNA expression in the midbrain (0.46 ± 0.02, P<0.05), compared with vehicle treated rats (0.75 ± 0.11). By contrast, FAAH protein levels in the midbrain were unaltered by URB597 treatment. FAAH mRNA and protein were unaltered in the liver of URB597-treated rats, compared to controls. NAPE-PLD and MAGL mRNA and protein levels were unaltered in the midbrain or liver of URB597-treated rats, compared to vehicle-treated rats. Following repeated URB597 treatment, the marked inhibition of FAAH activity in both the liver and midbrain is consistent with the elevation of FAEs in these tissues. The decrease in FAAH mRNA in the midbrain suggests that sustained inhibition of FAAH is associated with adaptive changes in the gene expression of FAAH, but not the other enzymes involved in endocannabinoid turnover. The Effect of Chemotherapy on Memory and Neurogenesis in Rats Laura Lyons, Geoff Bennett and Peter Wigmore, School of Biomedical Sciences, University of Nottingham Background: “Chemobrain” refers to the cognitive deficits some cancer patients experience after having undergone chemotherapy. There is evidence to suggest that cytotoxic drugs can impair the proliferation of neurones in the mature hippocampus, a process known as adult neurogenesis, which subsequently reduces ability to form new memories. Previous patient based studies have been limited by many confounding factors and we are currently investigating individual chemotherapeutical drugs. This study looks at cyclophosphamide (CP), a chemotherapeutic drug used to treat many cancers. It is able to cross the blood-brain barrier and we wish to investigate their effect on both cognitive performance and cellular changes in the hippocampus.
Results: Student's t-tests revealed both the drug treated and control group to retain the ability to distinguish an object in a novel location from that in a familiar one (p<0.05). No significant difference (p<0.05) was found between the quantities of doublecortin nor Ki67-positive cells in each group. Conclusions: The results indicate that CP has no short-term effect on hippocampal dependant memory formation or hippocampal neurogenesis. Future work within our laboratory hopes to identify drugs causing “chemobrain” and to prevent or counteract this deficit, using promoters of neurogenesis. MEMANTINE IMPROVES ISOLATION REARING-INDUCED RECOGNITION MEMORY DEFICITS IN RATS C. A. Jonesa, A.M. Browna, D.P. Auerb and K. C. F. Fonea aSchool of Biomedical Sciences; bAcademic Radiology; Institute of Neuroscience, Queen’s Medical Centre, University of Nottingham, Nottingham, U.K, NG7 2UH. Postweaning social isolation in rats is used as a neurodevelopmental model of schizophrenia, which induces several behavioural changes in adult rats resembling some of the positive, negative and cognitive symptoms of schizophrenia. We evaluated the effect of the uncompetitive NMDA receptor antagonist, memantine, on the cognitive and behavioural deficits induced by isolation-rearing to investigate whether glutamatergic dysfunction occurs in this paradigm. Male Lister-Hooded rats obtained immediately after weaning on postnatal day (PND) 24-25 were either group housed (3-4 per cage; n=9) or socially isolated (n=18) for a period of 6 weeks during which they received minimal handling but maintained visual, auditory and olfactory interaction with littermates. On PND’s 63, 70, 77 and 83 animals received either vehicle (2 ml/kg; i.p.) or memantine (10 or 15 mg/kg; i.p.) 20 mins prior to testing and novel cage induced locomotor activity (LMA), novel object recognition (NOR), prepulse inhibition (PPI) of acoustic startle and conditioned emotional response (CER) paradigms were evaluated respectively. Social isolation induced locomotor hyperactivity and NOR, PPI and CFC deficits in vehicle treated animals compared to group housed controls. Memantine significantly (p≤0.0001) suppressed the total LMA counts over 60 mins of socially isolated animals compared to group housed and isolation-reared controls. Group housed rats successfully discriminated between novel and familiar objects in the NOR task, whilst socially isolated rats explored both objects equally. This NOR deficit was significantly (p≤0.001) restored by memantine. Memantine failed to reverse either PPI or CFC impairments in isolation reared animals at the doses tested. The acute administration of memantine can therefore reverse isolation rearing-induced deficits in novel object recognition and suggests that glutamatergic dysfunction may contribute to the cognitive deficits seen in this animal model of schizophrenia. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||