Higher content of insulin-like growth factor-I in dystrophic mdx mouse: potential role in the spontaneous regeneration through an electrophysiological investigation of muscle function.

Insulin-like growth factor-I (IGF-I) is known to promote proliferation and differentiation of muscle cells during growth and regeneration. Both these conditions are characterized by acquisition of specialized muscle functions, such as a large macroscopic chloride conductance (GCl), a parameter that is a target of growth hormone (GH)/IGF-I axis action on skeletal muscle. The present study has been aimed at evaluating the role of IGF-I in the spontaneous regeneration occurring in hind limb muscle of dystrophic mdx mouse. IGF-I levels have been measured in hind limb muscles, plasma and liver of mdx and control mice of 8-10 weeks and 5 months of age by radioimmunoassay. In parallel the biophysical and pharmacological properties of muscle chloride channels of extensor digitorum longus (EDL) muscle fibers of mice belonging to the same age-group have been measured electrophysiologically in vitro. At 8-10 weeks of age, significantly greater amounts of IGF-I were found in plasma and hind limb muscles of mdx mice with respect to controls. Such a difference was only just detectable and no longer statistically significant at 5 months of age. No differences were found in hepatic IGF-I levels at either age. The EDL muscle fibers of mdx mice at 8-10 weeks of age were characterized by higher GCl values and by a different pharmacological sensitivity to the enantiomers of 2-(p-chlorophenoxy)-propionic acid (CPP), specific chloride channel ligands, with respect to age-matched controls. However, these differences were no longer detected at 5 months of age. Our results suggest a role of IGF-I in the high regenerative potential of muscles from mdx mice and support the hypothesis that the biophysical and pharmacological properties of chloride channels of EDL muscle fibers are sensitive indices of the action of regeneration-promoting factors on muscle function.

Phosphorylation and IGF-1-mediated dephosphorylation pathways control the activity and the pharmacological properties of skeletal muscle chloride channels.

1. In the present study we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates resting chloride conductance (G(Cl)) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating-dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2-(p-chlorophenoxy) propionic acid (CPP). 2. For this purpose G(Cl) in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4-beta-phorbol 12,13 dibutyrate (4-beta-PDB), presumably activating protein kinase C (PKC). The effects of IGF-1 and of the enantiomers of CPP on G(Cl) were then tested. 3. IGF-1 (3.3 nM) had no effect of G(Cl) on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of G(Cl) induced by 4-beta-PDB. No effects of IGF-1 were observed on G(Cl) lowered by the phosphatase inhibitor okadaic acid (0.25 microM). 4. Ceramide, reported to activate on okadaic acid-sensitive phosphatase, mimicked the effects of IGF-1. In fact, N-acetyl-sphingosine (2.5-5 microM), not very effective in control conditions, increased the G(Cl) lowered by the phorbol ester, but not the G(Cl) lowered by okadaic acid. 5. In the presence of 4-beta-PDB, G(Cl) was differently affected by the enantiomers of CPP. The S(-)-CPP was remarkably less potent in producing the concentration-dependent reduction of G(Cl), whereas the R(+)-CPP caused an increase of G(Cl) at all the concentrations tested. 6. In conclusion, the PKC-induced lowering of G(Cl) is counteracted by IGF-1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands.

Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibres of dystrophic mdx mouse and potential efficacy of taurine.

No clear data is available about functional alterations in the calcium-dependent excitation-contraction (e-c) coupling mechanism of dystrophin-deficient muscle of mdx mice. By means of the intracellular microelectrode "point" voltage clamp method, we measured the voltage threshold for contraction (mechanical threshold; MT) in intact extensor digitorum longus (EDL) muscle fibres of dystrophic mdx mouse of two different ages: 8 - 12 weeks, during the active regeneration of hind limb muscles, and 6 - 8 months, when regeneration is complete. The EDL muscle fibres of 8 - 12-week-old wildtype animals had a more negative rheobase voltage (potential of equilibrium for contraction- and relaxation-related calcium movements) with respect to control mice of 6 - 8 months. However, at both ages, the EDL muscle fibres of mdx mice contracted at more negative potentials with respect to age-matched controls and had markedly slower time constants to reach the rheobase. The in vitro application of 60 mM taurine, whose normally high intracellular muscle levels play a role in e-c coupling, was without effect on 6 - 8-month-old wildtype EDL muscle, while it significantly ameliorated the MT of mdx mouse. HPLC determination of taurine content at 6 - 8 months showed a significant 140% rise of plasma taurine levels and a clear trend toward a decrease in amino acid levels in hind limb muscles, brain and heart, suggesting a tissue difficulty in retaining appropriate levels of the amino acid. The data is consistent with a permanent alteration of e-c coupling in mdx EDL muscle fibres. The alteration could be related to the proposed increase in intracellular calcium, and can be ameliorated by taurine, suggesting a potential therapeutic role of the amino acid.

Effects of HMG-CoA reductase inhibitors on excitation-contraction coupling of rat skeletal muscle.

3-Hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors currently used as cholesterol-lowering drugs produce side effects in patients, one of which is myopathy. In the present study we compared the effect of a 3-month chronic treatment with two different compounds, simvastatin and pravastatin, on the excitation-contraction coupling of rat skeletal muscle fibers, the mechanism which links membrane depolarization to the movements of cytosolic Ca2+ from intracellular stores. The voltage threshold for mechanical activation of extensor digitorum longus muscle fibers in response to depolarizing pulses of various durations was studied in vitro by the two intracellular microelectrode method in 'point' voltage clamp mode. Simvastatin (5-50 mg/kg) modified the mechanical threshold of striated fibers in a dose-dependent manner. The muscle fibers of rats treated with 10 mg/kg and 50 mg/kg of simvastatin needed significantly less depolarization to contract than did untreated fibers at each pulse duration, suggesting that levels of cytosolic Ca2+ were higher. Consequently, the rheobase voltage for fiber contraction was significantly shifted toward more negative potentials with respect to controls by 2.4 mV and 7.1 mV in the 10 mg/kg and 50 mg/kg simvastatin-treated animals, respectively. Pravastatin treatment at 100 mg/kg did not produce any alteration of excitation-contraction coupling since the rheobase voltage was similar to that of controls. The different physicochemical properties of the two drugs may underlie the different effect observed because lipophilic agents, such as simvastatin, have been shown to affect sterol biosynthesis in many tissues, whereas the hydrophilic pravastatin is hepato-selective.

Hydroxyapatite coated with heaptocyte growth factor (HGF) stimulates human osteoblasts in vitro.

We have studied in vitro the effect of a hydroxyapatite (HA) tricalcium phosphate material coated with hepatocyte growth factor (HA-HGF) on cell growth, collagen synthesis and secretion of metalloproteinases (MMPs) by human osteoblasts. Cell proliferation was stimulated when osteoblasts were incubated with untreated HA and was further increased after exposure to HA-HGF. The uptake of [3H]-proline was increased after treatment with HA. When osteoblasts were exposed to HA-HGF, collagen synthesis was increased with respect to HA. The secretion of MMPs in control cells was undetectable, but in HA and HA-HGF cells MMP 2 and MMP 9 were clearly synthesised. Our results suggest that HA can promote osteoblast activity and that HGF can further increase its bioactivity.

Central depletion of brain-derived neurotrophic factor in mice results in high bone mass and metabolic phenotype.

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal differentiation/survival, the regulation of food intake, and the pathobiology of obesity and type 2 diabetes mellitus. BDNF and its receptor are expressed in osteoblasts and chondrocyte. BDNF in vitro has a positive effect on bone; whether central BDNF affects bone mass in vivo is not known. We therefore examined bone mass and energy use in brain-targeted BDNF conditional knockout mice (Bdnf(2lox/2lox)/93). The deletion of BDNF in the brain led to a metabolic phenotype characterized by hyperphagia, obesity, and increased abdominal white adipose tissue. Central BDNF deletion produces a marked skeletal phenotype characterized by increased femur length, elevated whole bone mineral density, and bone mineral content. The skeletal changes are developmentally regulated and appear concurrently with the metabolic phenotype, suggesting that the metabolic and skeletal actions of BDNF are linked. The increased bone development is evident in both the cortical and trabecular regions. Compared with control, Bdnf(2lox/2lox)/93 mice show greater trabecular bone volume (+50% for distal femur, P < 0.001; +35% for vertebral body, P < 0.001) and midfemoral cortical thickness (+11 to 17%, P < 0.05), measured at 3 and 6 months of age. The skeletal and metabolic phenotypes were gender dependent, with female being more affected than male mice. However, uncoupling protein-1 expression in brown fat, a marker of sympathetic tone, was not different between genotypes. We show that deletion of central BDNF expression in mice results in increased bone mass and white adipose tissue, with no significant changes in sympathetic signaling or peripheral serotonin, associated with hyperphagia, obesity, and leptin resistance.

Role of tumour necrosis factor alpha, but not of cyclo-oxygenase-2-derived eicosanoids, on functional and morphological indices of dystrophic progression in mdx mice: a pharmacological approach.

The role of tumour necrosis factor (TNF)-alpha or cyclo-oxygenase-2 (COX-2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4-8 weeks) adult dystrophic mdx mice with the anti-TNF-alpha etanercept (0.5 mg/kg) or the COX-2 inhibitor meloxicam (0.2 mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF-alpha by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise-induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept-treated muscles showed a reduction of connective tissue area and of pro-fibrotic cytokine TGF-beta1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4-week treatment was performed on 2-week-old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF-alpha, but not of COX-2 products, in different phases of dystrophic progression. Anti-TNF-alpha drugs may be useful in combined therapies for Duchenne patients.

Chronic administration of taurine to aged rats improves the electrical and contractile properties of skeletal muscle fibers.

A reduction of resting chloride conductance (GCl) and a decrease of the voltage threshold for contraction are observed during aging in rat skeletal muscle. The above alterations are also observed in muscle of adult rat after taurine depletion. As lower levels of taurine were found by others in aged rats compared to young rats, we tested the hypothesis that a depletion of taurine may contribute to the alteration of the electrical and contractile properties we found in skeletal muscle during aging. This was accomplished by evaluating the potential benefit of a pharmacological treatment with the amino acid. To this aim 25-mo-old Wistar rats were chronically treated (2-3 mo) with taurine (1 g/kg p.o. daily) and the effects of such a treatment were evaluated in vitro on the passive and active membrane electrical properties of extensor digitorum longus muscle fibers by means of current-clamp intracellular microelectrode technique. Excitation-contraction coupling was also evaluated by measuring the voltage threshold for contraction with the intracellular microelectrode "point" voltage clamp method. In parallel muscle and blood taurine contents were determined by high-performance liquid chromatography. Taurine supplementation significantly raised taurine content in muscle toward that found in adult rats. Supplementation also significantly increased GCl vs. the adult value, in parallel the excitability characteristics (threshold current and latency) related to this parameter were ameliorated. The increase of GCl induced by taurine was accompanied by a restoration of the pharmacological sensitivity to the R(+) enantiomer of 2-(p-chlorophenoxy) propionic acid, a specific chloride channel ligand. In parallel also the protein kinase C-mediated modulation of the channel was restored; in fact the potency of 4-beta-phorbol 12, 13-dibutyrate in reducing GCl was lower in taurine-treated muscles vs. untreated aged, being rather similar to that observed in adult. The treatment also improved the mechanical threshold for contraction of striated fibers which in aged rats is shifted toward more negative potentials, moving it toward the adult values. Our results suggest that the reduction of taurine content could play a role in the alteration of electrical and contractile properties observed during aging. These findings may indicate a potential application of taurine in ensuring normal muscle function in the elderly.

Protein kinase C antagonizes pertussis-toxin-sensitive coupling of the calcitonin receptor to adenylyl cyclase.

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis-toxin-sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin-dependent activation of adenylyl cyclase in HEK-293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor-dependent activation of Gi. The possibility that protein kinase C could be antagonizing Galphai-adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin-induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK-293 cells, that stably express a myc-tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Galphai subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Galphai is negatively regulated by protein kinase C.