Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism

The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.

Rho and smooth muscle cell phenotype

Smooth muscle cell (SMC) phenotypic modulation from the mature ’contractile’ to a less differentiated ’synthetic’ phenotype involves not only altered expression but also a reorganisation of contractile and cytoskeletal proteins. Objective: To investigate the role of RhoA, a known regulator of the actin cytoskeleton, in SMC phenotypic regulation. Methods: Rho transcription (RT-PCR), expression (Western analysis) and activation (membrane translocation or Rho ’pull-down’ assay) was investigated in cultured rabbit aortic SMC during phenotypic modulation, and under the influence of known SM-regulatory proteins (thrombin, heparin and TGF- β). Rho’s effect on cell morphology was examined by transient transfection of ’synthetic’ state SMC with either constitutively active Rho (Val14RhoA) or its inhibitor, C3 transferase. Results: RhoA transcription was elevated in the first 3 days of primary culture, and protein expression peaked at 2 days post-confluence when SMC return to a more ’contractile’ state. However, RhoA showed augmented activation at three time-points in primary culture: the transition point when SMCs enter logarithmic growth and are highly motile, upon reaching quiescence, and when they return to a more ’contractile’ state. Thrombin, heparin and TGF-β activated RhoA in ’synthetic’ state SMCs. Transfection with Val14RhoA caused a dramatic decrease in SMC size and a reorganization of cytoskeletal proteins, reminiscent of the ’contractile’ phenotype. Specific inhibition of endogenous Rho by C3 transferase resulted in an almost complete loss of contractile proteins. Conclusion: These data indicate that Rho is an important determining factor of SMC functional state.

The relative benefits of endurance and strength training on the metabolic factors and muscle function of people with type 2 diabetes mellitus

Objective: To compare the effects of a 4-month strength training (ST) versus aerobic endurance training (ET) program on metabolic control, muscle strength, and cardiovascular endurance in subjects with type 2 diabetes mellitus (T2D). Design: Randomized controlled trial. Setting: Large public tertiary hospital. Participants: Twenty-two T21) participants (I I men, I I women; mean age +/- standard error, 56.2 +/- 1.1 y; diabetes duration, 8.8 +/- 3.5y) were randomized into a 4-month ST program and 17 T2D participants (9 men, 8 women; mean age, 57.9 +/- 1.4y; diabetes duration, 9.2 +/- 1.7y) into a 4-month ET program. Interventions: ST (up to 6 sets per muscle group per week) and ET (with an intensity of maximal oxygen consumption of 60% and a volume beginning at 15min and advancing to a maximum of 30min 3X/wk) for 4 months. Main Outcome Measures: Laboratory tests included determinations of blood glucose, glycosylated hemoglobin (Hb A(1c)), insulin, and lipid assays. Results: A significant decline in Hb A, was only observed in the ST group (8.3% +/- 1.7% to 7.1% +/- 0.2%, P=.001). Blood glucose (204 +/- 16mg/dL to 147 +/- 8mg/dL, P <.001) and insulin resistance (9.11 +/- 1.51 to 7.15 +/- 1.15, P=.04) improved significantly in the ST group, whereas no significant changes were observed in the ET group. Baseline levels of total cholesterol (207 +/- 8mg/dL to 184 +/- 7mg/dL, P <.001), low-density lipoprotein cholesterol (120 +/- 8mg/dL to 106 +/- 8mg/dL, P=.001), and triglyceride levels (229 +/- 25mg/dL to 150 +/- 15mg/dL, P=.001) were significantly reduced and high-density lipoprotein cholesterol (43 +/- 3mg/dL to 48 +/- 2mg/dL, P=.004) was significantly increased in the ST group; in contrast, no such changes were seen in the ET group. Conclusions: ST was more effective than ET in improving glycemic control. With the added advantage of an improved lipid profile, we conclude that ST may play an important role in the treatment of T2D.

The orphan nuclear receptor, NOR-1, is a target of beta-adrenergic signaling in skeletal muscle

beta-Adrenergic receptor (beta-AR) agonists induce Nur77 mRNA expression in the C2C12 skeletal muscle cell culture model and elicit skeletal muscle hypertrophy. We previously demonstrated that Nur77 (NR4A1) is involved in lipolysis and gene expression associated with the regulation of lipid homeostasis. Subsequently it was demonstrated by another group that beta-AR agonists and cold exposure-induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77(-/-) animal model. These studies underscored the importance of understanding the role of NOR-1 in skeletal muscle. In this context we observed 30-480 min of beta-AR agonist treatment significantly and transiently increased expression of the orphan nuclear receptor NOR-1 in both mouse skeletal muscle tissue (plantaris) and C2C12 skeletal muscle cells. Specific beta(2)-and beta(3)-AR agonists had similar effects as the pan-agonist and were blocked by the beta-AR antagonist propranolol. Moreover, in agreement with these observations, isoprenaline also significantly increased the activity of the NOR-1 promoter. Stable exogenous expression of a NOR-1 small interfering RNA (but not the negative control small interfering RNA) in skeletal muscle cells significantly repressed endogenous NOR-1 mRNA expression and led to changes in the expression of genes involved in the control of lipid use and muscle mass underscored by a dramatic increase in myostatin mRNA expression. Concordantly the myostatin promoter was repressed by NOR-1 expression. In conclusion, NOR-1 is highly responsive to beta-adrenergic signaling and regulates the expression of genes controlling fatty acid use and muscle mass.

A role for Rho in smooth muscle phenotypic regulation

The role of the small GTP-binding protein Rho in the process of smooth muscle cell (SMC) phenotypic modulation was investigated using cultured rabbit aortic SMCs. Both Rho transcription and Rho protein expression were high for the first 3 days of culture (contractile state cells), with expression decreasing after change to the synthetic state and peaking upon return to the contractile phenotype. Activation of Rho (indicated by translocation to the membrane) also peaked upon return to the contractile state and was low in synthetic state SMCs. Transient transfection of synthetic state rabbit SMCs with constitutively active Rho (val14rho) caused a dramatic decrease in cell size and reorganization of cytoskeletal proteins to resemble those of the contractile phenotype; alpha-actin and myosin adopted a tightly packed, highly organized arrangement, whereas vimentin localized to the immediate perinuclear region and focal adhesions were enlarged. Conversely, specific inhibition of endogenous Rho, by expression of C3 transferase, resulted in the complete loss of actin and myosin filaments without affecting the distribution of vimentin. Focal adhesions were reduced in number. Thus, Rho plays a key role in regulating SMC phenotypic expression.

Homocysteine and cardiovascular disease in renal disease

Elevated homocysteine (hyperhomocysteinaemia) in renal patients is a major concern for physicians. Although cause and effect between homocysteine and cardiovascular disease (CVD) has not been established in either the general population or renal patients, there is much evidence that this relationship does exist. Purported mechanisms that may explain this effect include increases in endothelial injury, smooth muscle cell proliferation, low-density lipoprotein oxidation and changes in haemostatic balance. Renal patients have a much greater incidence of hyperhomocysteinaemia and this may be explained by decreases in either the renal or extrarenal metabolism of the compound. We conclude that data from long-term placebo-controlled trials are urgently required to determine whether hyperhomocysteinaemia in renal patients is a cause of CVD events and requires therapeutic targeting.

Emerging roles of urotensin-II in cardiovascular disease

Urotensin-II (UII) is a highly potent endogenous peptide within the cardiovascular system. Through stimulation of Galphaq-coupled UT receptors, UII mediates contraction of vascular smooth muscle and endothelial-dependent vasorelaxation, and positive inotropy in human right atrium and ventricle. A pathogenic role of the UT receptor system is emerging in cardiovascular disease states, with evidence for upregulation of the UT receptor system in patients with congestive heart failure (CHF), pulmonary hypertension, cirrhosis and portal hypertension, and chronic renal failure. In vitro and in vivo studies show that under pathophysiological conditions, UII might contribute to cardiomyocyte hypertrophy, extracellular matrix production, enhanced vasoconstriction, vascular smooth muscle cell hyperplasia, and endothelial cell hyper-permeability. Single nucleotide polymorphisms of the UII gene may also impart a genetic predisposition of patients to diabetes. Therefore, the UT receptor system is a potential therapeutic target in the treatment of cardiac, pulmonary, and renal diseases. UT receptor antagonists are currently being developed to prevent and/or reverse the effects of over-activated UT receptors by the endogenous ligand. This review describes UII peptide and converting enzymes, and UT receptors in the cardiovascular system, focusing on pathophysiological roles of UII in the heart and blood vessels. (C) 2004 Elsevier Inc. All rights reserved,

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Local therapy for restenosis

The durability of all forms of open or percutaneous revascularisation is affected by the development of localised stenoses within the bypass graft or at the site of endarterectomy, stent or angioplasty. The reported incidence of significant restenosis has varied dependent on initial procedure, site, case mix and definition, but is greatest during the first 12 months (Table 1).1 Over the last 40 years tens of thousands of studies have been carried out in an effort to understand or reduce the incidence of restenosis, with two major mechanisms identified as being responsible for the luminal narrowing, namely intimal hyperplasia and constrictive remodelling. Intimal hyperplasia is provoked by changes in the balance of local cytokines controlling vascular smooth muscle cell (VSMC) proliferation, apoptosis and migration, brought about by endothelial or medial injury and alterations in haemodynamic forces. The overall vessel diameter reduction that occurs in constrictive remodelling is less well defined, but likely involves matrix turnover under the control of proteinases, particularly metalloproteinases.

Retinal serotonin, eye growth and myopia development in chick

Myopia (short-sightedness) is a visual problem associated with excessive eye growth and vitreous chamber expansion. Within the eye serotonin (5-hydroxytryptamine, 5-HT) appears to have a variety of effects, it alters retinal amacrine cell processing, increases intraocular pressure, constricts ocular blood vessels, and is also mitogenic. This study sought to determine the role of the retinal serotonin system in eye growth regulation. Myopia was produced in 7-day-old chicks using -15 D spectacle lenses (LIM) and form deprivation (FDM). The effect on LIM and FDM of daily intravitreal injections of a combination of 5-HT receptor antagonists (1, 10, 50 mu M), 5-HT2 selective antagonist (Mianserin 0.5, 20 mu M) were assessed. Counts were performed of serotonin and tyrosine hydroxylase positive neurons and the relative density used to account for areal changes due to eye growth. The effect of LIM and lens-induced hyperopia (LIH) on the numbers of 5-HT-containing amacrine cells in the retina were then determined. The combination of the 5-HT receptor antagonists inhibited LIM by approximately half (1 mu M RE: -7.12 +/- 1.0 D, AL: 0.38 +/- 0.06 mm vs. saline RE: -13.19 +/- 0.65 D, AL: 0.64 +/- 0.03 mm. RE: p < 0.01, AL: p < 0.01), whereas FDM was not affected (1 mu M RE: -8.88 +/- 1.10 D). These data suggest that serotonin has a stimulatory role in LIM, although high doses of serotonin were inhibitory (1 mu M RE: -9.30 +/- 1.34 D). 5-HT immunoreactivity was localised to a subset of amacrine cell bodies in the inner nuclear layer of the retina, and to two synaptic strata in the inner plexiform layer. LIM eyes had increased numbers of 5-HT-containing amacrine cells in the central retina (12.5%). Collectively, these results suggest that manipulations to the serotonin system can alter the eye growth process but the role of the transmitter system within this process remains unclear. (c) 2005 Elsevier Ltd. All rights reserved.

Agonists and antagonists of protease activated receptors (PARs)

Protease activated receptors (PARs) are a category of G-protein coupled receptors (GPCRs) implicated in the progression of a wide range of diseases, including thrombosis, inflammatory disorders, and proliferative diseases. Signal transduction via PARs proceeds via an unusual activation mechanism. Instead of being activated through direct interaction with an extracellular signal like most GPCRs. they are self-activated following cleavage of their extracellular N-terminus by serine proteases to generate a new receptor N-terminus that acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. Short synthetic peptides corresponding to this newly exposed N-terminal tethered ligand can activate three of the four known PARs in the absence of proteases. and such PAR activating peptides (PAR-APs) have served as templates for agonist/antagonist development. In fact much of the evidence for involvement of PARs in diseases has relied upon use of PAR-APs. often of low potency and uncertain selectivity. This review summarizes current structures of PAR agonists and antagonists, the need for more selective and more potent PAR ligands that activate or antagonize this intriguing class of receptors, and outlines the background relevant to PAR activation, assay methods, and physiological properties anticipated for PAR ligands.