eIF3f depletion impedes mouse embryonic development,
reduces adult skeletal muscle mass and amplifies muscle
loss during disuse

Aurélie Docquier, Laura Pavlin, Audrey Raibon, Christelle Bertrand,
Chamroeun Sar, Serge S. Leibovitch, Robin Candau, Florence Sabatier, Henri
Bernardi

► To cite this version:

Aurélie Docquier, Laura Pavlin, Audrey Raibon, Christelle Bertrand, Chamroeun Sar, et al.. eIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse. 5. International Congress of Translational Research in Human Nutrition (ICTRNH 2019), Jun 2019, Clermont-Ferrand, France. hal-02737500

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Submitted on 2 Jun 2020

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# eIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse

Aurélie Docquierª, Laura Pavlinª, Audrey Raibonª, Christelle Bertrand-Gadayª, Chamroeun Sarᵇ, Serge Leibovitchª, Robin Candauª, Florence Sabatierª, Henri Bernardiª

## INTRODUCTION

Muscle mass homeostasis is controlled by protein synthesis and degradation. Eukaryotic initiation factor 3f, a subunit of eIF3 complex, is a fundamental element of MTORC1 pathway, allowing physical interaction between S6K1 and MTOR. Phosphorylation of downstream effectors of MTORC1 pathway like S6 leads to enhanced translational capacity and myotubes hypertrophy. Despite its important role in protein synthesis regulation shown in vitro, its physiological role in skeletal muscle is poorly described. Generation of mice carrying a mutation in the eIF3f gene allows to further define in vivo eIF3f roles in muscular homeostasis.

## MATERIAL & METHODS

Entire eIF3f allele coding sequence was substituted by *lacZ* reporter gene to generate heterozygous mice (eIF3f +/−). eIF3f +/− mice intercrosses were done to generate homozygous eIF3f knockout. Genotyping using eIF3f and *lacZ* primers allowed to identify newborn mutants. eIF3f mRNA and protein expression were analyzed in skeletal muscles of eIF3f +/− and wild-type (WT) mice. Then, 6/9-months-old C57BL/6 WT and eIF3f +/− males mice were submitted to unilateral immobilization for 3, 7 or 14 days. Mice were anesthetized by isoflurane inhalation to gently fix adhesive bandage. A puromycin injection was performed 15 minutes before cervical dislocation. Analysis of mass, cross-sectional areas (CSA), synthesis flows and MTORC1 pathway activity were all conducted on quadriceps muscle. Opposed hindlimb was used as an internal control.

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## MODEL CHARACTERIZATION

Values are mean ± SEM

Significantly different at * : p<0.05, ** : p<0.01 or *** : p<0.001

<table><caption>Embryonic death occurs at early stage of development</caption><thead><tr><td>Embryo phenotype</td><td>Normal</td><td>Necrotic</td></tr></thead><tbody><tr><td>Percentage (n=116)</td><td>76,7</td><td>23,3</td></tr></tbody></table>

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Normal

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Necrotic

<table><caption>Any homozygous eIF3f +/− embryo was recovered</caption><thead><tr><td>Embryo genotype</td><td>+/+</td><td>+/-</td><td>??</td></tr></thead><tbody><tr><td>Percentage (n=116)</td><td>25,8</td><td>50,2</td><td>24,0</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>Reduced eIF3f expression mainly impacts skeletal muscles mass</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>eIF3f +/− are viable and fertile and display diminished body mass</figcaption></figure>

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<figure><img src="image_9.png" /><figcaption>eIF3f mRNA and protein expression levels are lower in eIF3f +/− skeletal muscles</figcaption></figure>

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<figure><img src="image_11.png" /><figcaption>Fiber size is smaller without any change in fiber number in skeletal muscles of eIF3f +/− mice</figcaption></figure>

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<figure><img src="image_16.png" /><figcaption>Protein synthesis activity and efficiency are altered in eIF3f +/− mice</figcaption></figure>

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## HINDLIMB IMMOBILIZATION STUDY

<figure><img src="image_23.png" /><figcaption>Laminin labelling</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>Induction of eIF3f expression is decreased in eIF3f +/− mice</figcaption></figure>

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<figure><img src="image_27.png" /><figcaption>Activities of S6K1 and S6 are reduced in eIF3f +/− mice</figcaption></figure>

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<figure><img src="image_30.png" /><figcaption>Reduced eIF3f expression level exacerbates protein synthesis decrease</figcaption></figure>

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## CONCLUSION

Embryonic death suggests an essential role of eukaryotic Initiation Factor 3f for mice development. Whole body partial depletion allows normal growth despite a body mass reduction without change in body size. Mass lowering concerns only lean mass, skeletal muscles of heterozygous mice displaying a smaller fiber size. eIF3f mRNA and protein expressions are reduced in muscles of eIF3f +/− mice, as well as protein synthesis and MTORC1 pathway activity. During immobilization, reduction of eIF3f induction results in a stronger atrophy, with a larger decrease in muscle mass and CSA in eIF3f +/− mice. Protein synthesis is also reduced and associated with a lower MTORC1 pathway activity in eIF3f +/− mice.

These results confirm *in vivo* the essential role of eIF3f in development, muscular growth and muscle mass homeostasis.

<footnote>ªINRA — UMR 866 — Dynamique Musculaire & Métabolisme — 2 Place Pierre Viala, 34 060 Montpellier</footnote>

<footnote>ᵇINSERM — U-583 — Institut des Neurosciences de Montpellier — 80 Avenue Augustin Fliche, 34 090 Montpellier</footnote>