Endoplasmic Reticulum Oxidative Stress Triggers Tgf-Beta-Dependent Muscle Dysfunction by Accelerating Ascorbic Acid Turnover

Endoplasmic reticulum (ER) and oxidative stress are two related phenomena that have important metabolic consequences. As many skeletal muscle diseases are triggered by oxidative stress, we explored the chain of events linking a hyperoxidized ER (which causes ER and oxidative stress) with skeletal muscle dysfunction. An unbiased exon expression array showed that the combined genetic modulation of the two master ER redox proteins, selenoprotein N (SEPN1) and endoplasmic oxidoreductin 1 (ERO1), led to an SEPN1-related myopathic phenotype due to excessive signalling of transforming growth factor (TGF)-beta. The increased TGF-beta activity in the genetic mutants was caused by accelerated turnover of the ER localized (anti-oxidant) ascorbic acid that affected collagen deposition in the extracellular matrix. In a mouse mutant of SEPN1, which is dependent on exogenous ascorbic acid, a limited intake of ascorbic acid revealed a myopathic phenotype as a consequence of an altered TGF-beta signalling. Indeed, systemic antagonism of TGF-beta re-established skeletal muscle function in SEPN1 mutant mice. In conclusion, this study sheds new light on the molecular mechanism of SEPN1-related myopathies and indicates that the TGF-beta/ERO1/ascorbic acid axis offers potential for their treatment.


Material and Methods
Cell culture C2C12 cells were cultured in DMEM supplemented with 25 mM glucose and 10% fetal calf serum (FCS). SEPN1 was knocked down as described in 1 .

Western blot analysis
Muscle protein lysates were prepared after the mechanic pulverization and homogenization in lysis buffer. Whole cell protein lysates and muscle protein lysates were prepared with RIPA lysis buffer containing 10mM Tris-HCL pH7.4, 30mM NaCl, 1mM EDTA, 1% Nonidet P-40 and protease inhibitors. For the detection of unfolded procollagen the NP-40 solubilized lysates was challenged with 0.5% SDS and centrifugated through a glycerol cushion. For the detection of puromycin, muscle protein lysates were prepared with 40mM Tris PH 7.5, 1mM EDTA, 5mM EGTA pH8.5 and 0.5% Triton and protease and phosphatase inhibitors. Protein concentration was determined for each sample by using the Bicinchoninic acid assay and equal amounts of proteins were loaded and resolved by 10% SDS-PAGE. Full list of antibodies used is provided in the paragraph Antibodies.

AAV injection
ERO1 and GFP-AAV2/1 vectors were produced by the TIGEM AAV Vector Core by triple transfection of HEK-293 cells followed by two rounds of CsCl 2 purification. For each viral preparation, physical titers (genome copies-GC/ml) were determined by averaging the titer achieved by dot-blot analysis and by PCR quantification using TaqMan (Applied Biosystems, Carlsbad, CA, USA).
One-month-old WT and SEPN1KO mice were anesthetized and injected with a total dose of 10 11 GC of ERO1a-AAV2/1 in three sites of the right gastrocnemius. Equivalent doses of GFP-AAV2/1 or equal volumes of PBS were injected into the contralateral muscle.
The animals were sacrificed 4 weeks after being injected and gastrocnemii, free from neighbouring muscles were isolated and analysed.

Real-time quantitative RT-PCR analysis
Total RNA was isolated from muscle tissues using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. One µg of total RNA was reversetranscribed and analyzed with the Applied Biosystems Real-time PCR System using the ΔΔCt method. Relative gene expression was normalized to GAPDH mRNA levels (as was the houskeeping gene with a lower coefficient of variance among the microarray data) and the graphs show the mean expression levels of the indicated genes in the muscles of the indicated genotipes relative to those of the WT muscles.
The Real time primers were as follows: gene was used as the nuclear marker gene to standardize mtDNA copy number to diploid chromosomal DNA content according to 2 .

Statistical comparison between Gulo and DKO
Most of the comparisons discussed in the paper are between DKO and Gulo KO mice fed with the same dose of ascorbic acid as these Gulo mice are the most appropriate reference. It could be possible to compare DKO with SEPN1KO mice too but, in the majority of cases, the latter do not show any alteration. A. The upper traces show a peak signal for the standard ascorbic acid that is not detected in the mobile phase indicating that the method is specific for the detection of ascorbic acid. The lower traces show a peak signal of Ascorbic acid in the gastrocnemius of the indicated genotypes, that is absent after treatment with the oxidant H 2 O 2 and is greatly reduced after one week of ascorbic acid starvation in Gulo KO mice.
B. Abundance of HSP47 mRNA relative to wt (fold change) measured by quantitative real time PCR in cDNA from gastrocnemii (n=3/5 per each group, bar graphs indicate means ±SEM, differences were examined using a 2-tailed unpaired Student's t tests and P values are indicated on the graph).
C. Representative histology of collagen I staining. The insets show the accumulation of collagen in DKO maintained at the low dose of ascorbic acid (scale bar: 100 mm).

Sup. Figure 4 related to Figure 6
A. Western blotting analysis of the rate of new protein synthesis in the gastrocnemii of mice with the indicated genotype and treatment. Puromycin incorporation was detected with anti-puromycin (anti-Pur) antibody, and GAPDH was used as loading control.
Graph shows the signal of puromycin of gastrocnemii of four different mice.
B. Real-time PCR quantification of the amount of mtDNA relative to that of RNAse-P, a nuclear gene used as a standard (n=7 per each group, bar graphs indicate means ±SEM, 7 differences were examined using a 2-tailed unpaired Student's t tests and P value is indicated on the graph).