Densitometric quantification was completed using ImageJ software. Statistical Analysis To test whether changes in gene expression levels were significant between fed and fasted mice, we used one-way ANOVA followed by Post-hoc tests using the Bonferroni correction for multiple testing.P-values lower than 0.05 were considered significant. == Results == Autophagy is impaired inmdxmice To investigate autophagy regulation inmdxmice, we chose the tibialis anterior and diaphragm muscles as they are examples of glycolytic and oxidative muscles, respectively19,27,41. impaired in the tibialis anterior muscle of mdx mice and that the regulation of autophagy is muscle type dependent. Differences between muscle groups should be considered during the pre-clinical development of therapeutic strategies addressing muscle metabolism. == Background == Duchenne Muscular Dystrophy (DMD) is the most common neuromuscular disorder. DMD is caused by the complete absence of the dystrophin protein, which leads to extensive muscle degeneration and regeneration followed by substitution of muscle with fibrotic and adipose tissues1,2. No cure is yet available, but several therapeutic approaches aiming at reversal of the ongoing degeneration have been investigated in preclinical and clinical settings with disappointing results3,4,5. Currently, drugs intended to induce skeletal muscle hypertrophy via Akt-mediated protein synthesis are in preclinical (e.g. valproic acid) or clinical (e.g. IGF-1) development6,7,8(see also http://clinicaltrials.gov/ct2/show/NCT01207908). IGF-1 is able to trigger Akt phosphorylation via class I PI3K9,10, which in turn induces a series of biochemical changes leading to protein synthesis via the mammalian target of rapamycin (mTOR) pathway11,12,13. At the same time, Akt is able to repress catabolic pathways, such as macroautophagy (hereafter referred as autophagy) and ubiquitin-proteasome, leading to muscle LY 344864 atrophy14,15. This repression can occur in a transcriptional and non-transcriptional fashion. Indeed, it is known that mTOR inhibition by rapamycin induces autophagy LY 344864 without affecting gene expression, while Akt can repress the transcription factor FoxO3. This transcription factor is involved in the transcriptional activation of the atrophy genesAtrogin1andMuRF1and the autophagy geneBnip316,17. Therefore, the autophagy-lysosome system might also be a potential target for therapeutic intervention for muscular dystrophies. The autophagic pathway is responsible for the removal of unfolded/toxic proteins as well as dysfunctional/abnormal organelles. It is constantly active in skeletal muscle and is involved in several conditions such as denervation, cachexia and fasting18. We recently reported that autophagy is impaired in collagen VI-related myopathies and that induction of autophagy can rescue myofibers defects of collagen VI deficient mice19,20. The Akt/mTOR axis is one of the key pathways regulating autophagy. Previous studies showed that Akt signaling is affected in dystrophin nullmdxmice21. In fact, Akt signaling is strongly enhanced in 4-week-oldmdxmice,i.e.the period when extensive muscle regeneration is occurring22. This enhancement of Akt signaling decreases over time with a slight up-regulation in 3-month-old mice23,24and hardly any up-regulation in older mice25. Recently it was reported that both activation of autophagy by an AMPK agonist and inhibition of autophagy by Akt activation via valproic acid could ameliorate the dystrophic phenotype ofmdxmice6,26. Furthermore, it was shown that autophagy is impaired in both glycolytic and oxidative muscles ofmdxmice27. In the present study, we analyzed the Akt/mTOR pathway under basal conditions and after fasting inmdxand wild-type mice. We found a persistent activation of the Akt/mTOR pathway after fasting in themdxmice tibialis anterior but not in the diaphragm. Taken together, these data show that abnormal Akt signaling differentially impacts the regulation of the autophagy machinery in diverse dystrophin deficient muscles. == Methods == Ethical approval All procedures were approved by the Animal Welfare Commission of the Leiden University Medical Center (work protocol 11071). The institution is authorized by the government to judge the proposals according to the law. All experiments were performed LY 344864 in accordance with the regulations for animal experimentation. Mice C57BL/10ScSn-mdx/J (mdx) and control C57BL/10 mice were fed Mouse monoclonal antibody to MECT1 / Torc1 ad libitum with chow until 16 weeks of age. At this age, mice were divided into the fed or fasting groups (4-5 mice per group). Fasting started at 9 am in the morning and lasted for 24 hours. Mice from both groups were then sacrificed by cervical dislocation. Tibialis anterior and diaphragm muscles were harvested and immediately frozen in liquid nitrogen before further processing. q-RT-PCR analysis Total RNA was isolated using Tripure reagent as described previously28. The RNA concentration was measured on a Nanodrop (Nanodrop Technologies, USA) and integrity was checked with a total RNA nano chip assay on the Agilent 2100 bioanalyzer (Agilent, the Netherlands). cDNA synthesis was performed using random hexamer primers and gene expression levels were determined by Sybr Green based Real Time qPCR on the Roche Lightcycler 480 (Roche Diagnostics Ltd, UK). All primer pairs used spanned at least one splice junction to avoid contamination with genomic DNA amplification. Relative expression was determined using Gapdh as reference gene, while primer efficiencies were determined with LinReg PCR version 11.3. Western Blot Frozen muscles were homogenized by grinding in.
