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Potential involvement of angiotensin II in pathological signs of dystrophic muscle: In vivo and ex vivo outcome of a chronic treatment with enalapril, an angiotensin-converting enzyme inhibitor, in the exercise-aggravated mdx mouse model
No full textDisease-Related Modification of Chloride Conductance in Skeletal Muscle of Dystrophic Mice: Expression of CLC-1 Channel and Role of Inflammation and Oxidative Stress-Related Signaling
No full textEvaluation of potential synergistic action of a combined treatment with alpha-methyl-prednisolone and taurine on the mdx mouse model of Duchenne muscular dystrophy.
No full textAims: Glucocorticoids are the sole drugs clinically used
in Duchenne muscular dystrophy, in spite of the relevant
side effects. Combination of glucocorticoids with synergistic
drugs may be one strategy to lower doses and
control side effects, meanwhile providing wider control
of the complex pathology. This study is a preclinical
evaluation of the effect of a combined treatment of amethyl-
prednisolone (PDN) with taurine, a safe aminoacid
with positive effects on some pathology-related
events. Methods: PDN (1 mg/kg/day i.p.) and taurine
(1 g/kg/day orally) were administered either alone or in
combination, for 4–8 weeks to male dystrophic mdx mice
chronically exercised on a treadmill. Effects were assessed
in vivo and ex vivo with a variety of methodological
approaches. Results: In vivo, each treatment significantly
increased fore limb strength, a marked synergistic effect
being observed with the combination PDN + taurine. Exvivo, PDN + taurine completely restored the mechanical
threshold, an electrophysiological index of calcium
homeostasis, of extensor digitorum longus myofibres and
the benefit was greater than for PDN alone. In parallel,
the overactivity of voltage-independent cation channels
in dystrophic myofibres was reduced. No effects were
observed on plasma levels of creatine kinase, while
lactate dehydrogenase was decreased by taurine and, to a
minor extent, by PDN + taurine. A similar histology
profile was observed in PDN and PDN + taurine-treated
muscles. PDN + taurine significantly increased taurine
level in fast-twitch muscle and brain, by high-pressure
liquid chromatography analysis. Conclusions: The combination
PDN + taurine has additive actions on in vivo
and ex vivo functional end points, with less evident
advantages on histopathology and biochemical markers
of the disease
Parallel effects of α-methyl-prednisolone on skeletal muscle and brain of mdx mice: Identification of a novel mechanism of action.
No full textGlucocorticoids are clinically used in Duchenne muscular dystrophy although their mechanism of action is largely unclear. Part of their effect in dystrophic muscle can be mediated by the enhancement of utrophin expression; however the impact of this increase on dystrophin–glycoprotein complex (DGC) is unknown. In the present work we assessed the effect of a chronic treatment with α-methyl-prednisolone (PDN) (4–6 weeks at 1 mg/kg day) in the exercised mdx mouse model, on the expression of utrophin and key proteins of the DGC in skeletal muscle. We found a significant increase in expression of utrophin, α and β dystroglycan and DGC-bound aquaporin (AQP)-4 at both mRNA and protein level in PDN-treated muscles. Interestingly, this was paralleled by a normalization of phosphorylation state of β-dystroglycan and an increase in laminin. Immunohistochemistry supported the correct sarcolemmal localization of the proteins. On pathology-related signs, PDN increased mdx mouse force in vivo, while ex vivo it ameliorated calcium homeostasis and reduced signs of myofiber degeneration; however plasma creatine kinase and susceptibility to eccentric contraction were not ameliorated. In parallel we also assessed the impact of PDN treatment on blood–brain barrier (BBB), based on its serious impairment in mdx mice and on the clinical use of glucocorticoids in disorders characterized by leaky BBB. Interestingly, we found a restoration of key BBB markers at level of endothelium (ZO-1 and occludin), pericytes (desmin) and glial cells (glial fibrillary acidic protein, GFAP) in PDN-treated mdx mice. In parallel, an increase of mRNA and protein content of DGC (α–β dystroglycan complex Dp 71, AQP4), and a rescue of the phosphorylation state of AQP4 and β dystroglycan were found. Then PDN exerts a positive action on DGC in both muscle and brain, disclosing a novel mechanism of action whose impact on therapeutic effect deserves to be better investigated (Supported by Duchenne Parent Project NL)
Enalapril treatment discloses an early role of angiotensin II in inflammation- and oxidative stress-related muscle damage in dystrophic mdx mice
No full textInhibitors of angiotensin converting enzymes (ACE) are clinically used to control cardiomyopathy in patients of Duchenne muscular dystrophy. Various evidences suggest potential usefulness of long-term treatment with ACE inhibitors to reduce advanced fibrosis of dystrophic muscle in the mdx mouse model. However, angiotensin II is known to exert pro-inflammatory and pro-oxidative actions that might contribute to early events of dystrophic muscle degeneration. The present study has been aimed at evaluating the effects of an early treatment with enalapril on the pathology signs of exercised mdx mouse model. The effects of 1 and 5 mg/kg enalapril i.p. for 4-8 weeks have been compared with those of 1 mg/kg α-methylprednisolone (PDN), as positive control. Enalapril caused a dose-dependent increase in fore limb strength, the highest dose leading to a recovery score similar to that observed with PDN. A dose-dependent reduction of superoxide anion production was observed by dihydroethidium staining in tibialis anterior muscle of enalapril-treated mice, approaching the effect observed with PND. In parallel, a significant reduction of the activated form of the pro-inflammatory Nuclear Factor-kB has been observed in gastrocnemious muscle. Histologically, 5 mg/kg enalapril reduced the area of muscle necrosis in both gastrocnemious muscle and diaphragm, without significant effect on non-muscle area. In parallel no significant changes have been observed in both muscle TGF-β1 and myonuclei positive to phosphorylated Smad2/3. Myofiber functional indices were also monitored by microelectrodes recordings. A dose-dependent recovery of macroscopic chloride conductance has been observed upon enalapril treatment in EDL muscle, with minor effects being exerted in diaphragm. However a modest effect, if any, was found on mechanical threshold, a functional index of calcium homeostasis. No recovery was observed in creatine kinase and lactate dehydrogenase. Finally the results suggest the ability of enalapril to blunt angiotensin-II dependent activation of pro-inflammatory and pro-oxidant pathways which may be earlier events with respect to the pro-fibrotic ones, and may in part account for both functional impairment and muscle necrosis. The PDN-like profile may corroborate the combined use of the two classes of drugs in DMD patients so to potentiate the beneficial effects at skeletal muscle level, while reducing both spontaneous and PDN-aggravated cardiomyopathy
Potential role of sirtuin-1 as druggable target in muscular dystrophy: effect of a chronic resveratrol treatment on in vivo and ex vivo pathological signs of dystrophic mdx mouse
No full textPharmacological evaluation of NADPH oxidase involvement in pathophysiology of mdx mouse, an animal model of muscular dystrophy
No full textOxidative stress, caused by reactive oxygen species (ROS), has been implicated on disease progression and chronic inflammation in Duchenne Muscular Dystrophy (DMD). NADPH oxidase 2 (NOX2) is currently considered to be a major source of ROS and it is over-expressed in skeletal muscle and heart of mdx mice, the most widely used model for DMD. Its activation in mdx myofibers via stretch-sensitive pathways has also been shown (Whitehead et al., 2010; Khairallah et al., 2012). Consequently, drugs able to reduce ROS production by inhibition of NOX-2 are potential treatment for muscular dystrophy. In line with this view, we have recently shown that enalapril, by inhibiting the production of angiotensin II (Ang II), one of the main endogenous activator of NOX, reduces the signs of oxidative stress and the percentage of p65-NF-kB positive nuclei in the mdx muscles. In this frame we also observed, in the myofibers of mdx mice treated with enalapril, a dose-dependent restoration of macroscopic chloride conductance (gCl), a sensitive biomarker of inflammation in skeletal muscle (Cozzoli et al., 2011). The aim of the present study was to investigate the involvement of NOX-2 dependent-ROS production in relation to the aberrant mechano-transduction occurring in dystrophic muscle. RT-PCR experiments confirmed a higher expression of β-tubulin and NOX2 (gp91phox) mRNA in gastrocnemius (GC) muscle of mdx mice. Interestingly, this increased expression was maintained in GC muscles of mdx mice that underwent a standard chronic (1-2 months) exercise protocol on treadmill. Then, we tested the effect of a chronic treatment with apocynin (38 mg/kg in drinking water/day for 5-9 weeks), a natural compound able to directly inhibit NOX-2, on exercised mdx mice (De Luca et al., 2003). Treatment started at 4-5 weeks of age and the outcome was evaluated by a multidisciplinary approach on pathology-related in vivo and ex vivo endpoints. In vivo, apocynin significantly increased mouse strength, with normalized forelimb force values of 6.4 ± 0.16 (n=9) vs. 5.6 ± 0.19 (n=10; p<0.05) of untreated mice, but did not improve exercise performance. Furthermore, no effect was observed on plasma creatine kinase and lactate dehydrogenase. However, the treatment with apocynin counteracted the exercise-induced impairment of total membrane conductance (gm), which is mainly sustained by the reduction of gCl, in extensor digitorum longus (EDL) muscle fibers, gm being 2536 ±105 μS/cm2 (n =37) vs. 1886 ± 92 μS/cm2 (n = 42, p <0.0001) of untreated mdx myofibers. Then the recovery score for this parameter, considering the value of 2607 ± 23 μS/cm2 (n =19) of wild-type C57BL10 myofibers, was 90% in apocynin-treated myofibers. This latter effect prompted us to investigate the possibility that the channels underlying macroscopic gCl could be target of the redox-dependent NOX actions in skeletal muscle. Parallel experiments on EDL muscle fibers of C57BL10 mouse showed that Ang II decreases gCl in a concentration-dependent manner (IC50 = 60nM) and this effect was fully contrasted by the prior incubation of apocynin (10 μM) or with a known anti-oxidant N-acetyl cysteine (5 mM). Markers of oxidative stress and inflammation, RT-PCR, histo-morphology are currently under evaluation. However, this preliminary data support the hypothesis that pharmacological targeting of NOX-2, providing protection from cross-talk between ROS production and inflammation, may represent a valuable approach in DMD. (Supported by DPP/NL and MIUR-PRIN n° 20108YB5W3).
Whitehead et al. (2010). PLoS One, 5(12):e15354.
Khairallah et al. (2012). Sci Signal.5(236):ra56.
Cozzoli et al. (2011). Pharmacol Res. 64(5):482-92.
De Luca et al. (2003) J Pharmacol Exp Ther.304(1):453-63
Toward the identification of druggable pathways involved in disease-related fatigue in Duchenne muscular dystrophy: In vivo and ex vivo studies in dystrophic mdx mice
No full textEvaluation of potential efficacy of GLPG0492, a novel selective androgen receptor modulator, in the exercised-mdx mouse model: Comparison with α-methyl prednisolone and nandrolone
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