Glucocorticoids improve myogenic differentiation in vitro by suppressing the synthesis of versican, a transitional matrix protein overexpressed in dystrophic skeletal muscles

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration

Hierarchical assembly of discrete copper(ii) metallo-structures from pre-assembled dinuclear (bis-beta-diketonato)metallocycles and flexible difunctional co-ligands

The sequential interaction of preformed [Cu(L) (THF)] (where HL is 1,1-(1,3-phenylene)-bis(4,4-dimethylpentane-1,3-dione incorporating a 1,3-phenylene linker between its two β-diketone domains) and [Cu (L)]·2HO (where H L is 1,1-(4,4′-oxybiphenylene)-bis(4,4- dimethylpentane-1,3-dione) incorporating a flexible oxybiphenylene linkage between the two β-diketone groups) with the potentially difunctional aliphatic non-planar co-ligands, N-methylpiperazine (mpip), N,N′- dimethylpiperazine (dmpip) and 1,4-thiomorpholine (thiomorph) is reported. A series of extended molecular assemblies exhibiting a range of di- and tetranuclear assemblies were obtained and their X-ray structures determined. Dinuclear [Cu(L)(mpip)] ·2mpip incorporates two 5-coordinate, square pyramidal metal centres as does tetranuclear [{Cu(L)} (dmpip)]·2dmpip. In contrast, dinuclear [Cu (L)(dmpip)]·dmpip and [{Cu(L)}(thiomorph) ]·3thiomorph each contain two 5-coordinate and two 6-coordinate centres. Each of [Cu(L)(THF) ]·2THF and Cu(L)(mpip) ]·HO incorporate only 5-coordinate metal centres, with the latter complex forming a one-dimensional hydrogen bonded ribbon-like structure directed along the crystallographic a-axis. In keeping with the documented tendency for the smallest, least strained assembly to form in supramolecular self-assembly processes, the incorporation of the flexible "oxy" linkage between the 4,4′-linked phenylene rings of H L results in generation of a dinuclear [Cu L] species rather than a trinuclear (triangular) [CuL] species of the type formed by the more rigid bis-β-diketonato ligand analogue in which the biphenylene rings separating the β-diketone domains are directly coupled in their 4,4′ positions

Gastrointestinal Physiology of Chinook Salmon, Oncorhynchus tshawytscha (Walbaum) with Gastric Dilation Air Sacculitis (GDAS)

The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated

Influence of Oxygen Supply on Metabolism and Energetics in FishMuscles

The five discrete, but related studies presented in this thesis investigate several aspects of the physiology and biochemistry of whole animals, perfused and isolated tissues from fishes and other vertebrates. Important fundamental questions about tissue metabolism and energy supply and utilisation in relation to oxygen supply, in addition to applied questions relating to commercial harvesting and post-mortem muscle physiology were addressed. Oxyconformance of oxygen consumption (VO2) at low oxygen delivery rates was shown using an isolated, perfused salmon tail preparation, composed primarily of skeletal muscle. Addition of pig red blood cells to the perfusing solution at a haematocrit of 5 or 10%, increasing the capacitance, resulted in oxyregulation of VO2 by the tail tissues. Below c.60 ml O2.kg-1.h-1 of oxygen delivery, VO2 was delivery dependent. Above this value additional oxygen delivery did not increase VO2 of resting muscle above c.35 ml O2 kg-1.h-1. The preparation was validated by measuring mitochondrial activity using MTT and blood flow distribution to the red and white muscle using fluorescent microspheres. Evidence of both O2-independence of VO2 in the vasculature and strict O2-dependence of VO2 in striated muscles of fishes and a mammal is presented using isolated vascular tissue and an in vitro tissue slice model. VO2 by vessels from rat, salmon and hagfish showed varying degrees of independence between PO2s of 15-95 mmHg in vitro (1 mmHg = 0.133 kPa). Above and below these values, VO2 was highly PO2-dependent. VO2 by cardiac and skeletal muscles from rat, salmon, snapper and hagfish were shown to relate linearly to PO2 between zero and 125 mmHg. VO2 in these tissues was highly dependent on tissue type (cardiac, red and white muscle) which correlated with haem protein concentration. The increase in VO2 in muscle slice mitochondria uncoupled with FCCP and DNP ruled out diffusion-limitation as a constraint on VO2. Mitochondrial activity was constant over time and reoxygenation of the Ringer bathing the tissues after the initial run down in PO2 resulted in VO2 rates that were unchanged from the starting values, demonstrating that the tissues remained viable over time. ATP turnover in red muscle was significantly increased at 100 mmHg relative to 30 mmHg, and increased in both treatments from values at the start. Our data suggest that ATP supply and ATP demand were reduced in conjunction with falling PO2. The effects of hydrogen sulphide (H2S) (derived from Na2S) and isoeugenol exposure on activity, VO2 and ventilation frequency (Vf) in a teleost fish are reported. In the H2S treatment group (200 μM Na2S) both resting VO2 and Vf decreased after 30 minutes of exposure, concurrent with narcosis and a loss of equilibrium. These events corresponded with a significant fall in VO2 (33%) and Vf (20%) by 15 minutes, both declining further to a nadir of 40% of resting values at 30 minutes. After flushing, VO2 increased to resting levels, with Vf remaining significantly depressed until 30 minutes of recovery. Recovery was accompanied by regained mobility and equilibrium and significantly increased VO2 and Vf. Isoeugenol anaesthetised fish (0.011 g.L-1) reached stage 4-5 of anaesthesia accompanied by significant decreases in VO2 (45%) and Vf (25%) at 25 minutes, both parameters declining further to around 64% and 38% respectively by 35 minutes. Similar to H2S exposed fish, VO2 increased to resting values after flushing, followed by a significant rise in VO2. Likewise, Vf had risen to resting values post-flushing, subsequently increasing significantly during recovery. Overall, VO2 in relation to resting rate was reduced in the isoeugenol treated animals, while in H2S treated fish, exposure there was increased oxygen usage, possibly associated with a toxic effect. H2S significantly reduced cytochrome c oxidase activity in muscle and gill tissue in vitro between 69-79% at 20 μM and 77-97% at 200 μM Na2S, while isoeugenol had no effect on activity in any tissue. Calorimetric and biochemical profiles of anoxic, post-mortem white muscle from Chinook salmon subjected to rested and exhausted harvesting regimens at their acclimation temperature (10°C) are reported. Prior to harvest rested animals were anaesthetised with 0.012 g.L-1 isoeugenol without disturbance. The muscle of these animals had a high metabolic rate at the time of death, at around 400 μW.g-1, which declined rapidly over the first 12 hours to15 μW.g-1. Exhausted animals were forced to swim and were crowded before capture, resulting in an initial heat output of <10 μW.g-1. Heat output was significantly greater in the rested group at the time of death and for 7 hours post-mortem. In both groups there was an exothermic event, occurring between 4 and 6 hours post-mortem amounting to a rise of around 35 μW.g-1. A one-phase exponential decay model appropriately described the net heat output of the rested profile minus the exhausted data. Rested animals had significantly higher initial cut surface pH (7.5 vs 6.7), tissue glycogen (16 vs 2 μmol.g-1), creatine phosphate (18 vs 0.1 μmol.g-1), ATP (6 vs 3.5 μmol.g-1) and potential energy (30 vs 7 μmol.g-1) than the exhausted group, which had significantly elevated tissue concentrations of lactate (43 vs 18 μmol.g-1) and glucose (5 vs 2 μmol.g-1). Potential energy in the form of ATP, glycogen and creatine phosphate remained elevated for an extended period post-mortem in rested animals while catabolites further down the chain such as inosine, hypoxanthine and uric acid accumulated at similar rates in both groups. We examined the relationship between exogenous and endogenous H2S and oxygen partial pressure in isolated hagfish and lamprey vessels that exhibit profound hypoxic vasoconstriction (HVC). In myography studies, H2S (Na2S) dose-dependently constricted dorsal aortas (DA) and efferent branchial arteries but did not affect ventral aortas or afferent branchial arteries, which was similar to the effects produced by hypoxia. Sensitivity of H2S-mediated contraction in hagfish and lamprey DA was enhanced by hypoxia. HVC in hagfish DA was enhanced by the H2S precursor cysteine and inhibited by amino-oxyacetate (AOA), an inhibitor of the H2S-synthesising enzyme, cystathionine β-synthase, and unaffected by propargyl glycine, an inhibitor of cystathionine λ-lyase. Oxygen consumption (MO2) of hagfish DA was constant between a PO2 of 15 and 115•mmHg, decreased when PO2 <15•mmHg, and increased if PO2 exceeded 115•mmHg. 10 μmol.l-1 H2S increased and concentrations above 100 μmol.l-1 H2S decreased MO2. Consistent with the effects on HVC, cysteine increased and AOA and hydroxylamine, an inhibitor of pyridoxyl 5’-phosphate-dependent enzymes, decreased MO2. These data show that H2S is a monophasic vasoconstrictor of specific cyclostome vessels and because hagfish lack vascular NO, and vascular sensitivity to H2S was enhanced at low PO2, it is unlikely that H2S contractions are mediated by either an H2S-NO interaction or an oxidation product of H2S. These experiments provide additional support for the hypothesis that the metabolism of H2S is involved in oxygen sensing/signal transduction in vertebrate vascular smooth muscle. Together the findings of this thesis contribute to the understanding of oxygen utilisation and energetics in relation to oxygen supply in a number of tissues. These data further our understanding of respiratory physiology and may have practical applications in the seafood industry

Oxygen dependence of metabolic rate in the muscles of craniates

We present evidence that oxygen consumption (VO2 ) is oxygen partial pressure (PO2) dependent in striated muscles and PO2 -independent in the vasculature in representatives of three craniate taxa: two teleost fish, a hagfish and a rat. Blood vessel VO2 displayed varying degrees of independence in a PO2 range of 15&ndash;95 mmHg, while VO2 by striated muscle tissue slices from all species related linearly to PO2 between 0 and 125 mmHg, despite VO2 rates varying greatly between species and muscle type. In salmon red muscle, lactate concentrations fell in slices incubated at a PO2 of either 30 or 100 mmHg, suggesting aerobic rather than anaerobic metabolism. Consistent with this finding, potential energy, a proxy of ATP turnover, was PO2 -dependent. Our data suggest that the reduction in VO2 with falling PO2 results in a decrease in ATP demand, suggesting that the hypoxic signal is sensed and cellular changes effected. Viability and diffusion limitation of the preparations were investigated using salmon cardiac and skeletal muscles. Following the initial PO2 depletion, reoxygenation of the Ringer bathing salmon cardiac muscle resulted in VO2s that was unchanged from the first run. VO2 increased in all muscles uncoupled with p-trifluoromethoxylphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP). Mitochondrial succinate dehydrogenase activity, quantified by reduction of 3-(4,5-dimethylthiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, was constant over the course of the experiment. These three findings indicate that the tissues remained viable over time and ruled out diffusion-limitation as a constraint on VO2.<br /

Characterisation and vascular expression of nitric oxide synthase 3 in amphibians

In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81&nbsp;% sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3