Effect of leucine on the expression of miR-299a and HDAC4 in the skeletal muscle of rats submitted to hind limb immobilization: implications for the control of muscle mass

Abstract

This project was conceived from the thematic project FAPESP 2015 / 04090-0, in which the analysis of an RNAseq of the soleus muscle of immobilized rats and supplemented with leucine identified 47 differentially expressed genes, modulated by immobilization and attenuated by leucine, among the which HDAC4 (Histone Deacetylase 4) stood out for its responsiveness (for reference, we forward the work published by Alves et al., 2020; doi: 10.3390/cells9122582, in annex). Thus, the directly linked Ph.D. fellowship (FAPESP 2018/24419-4) aims to understand the anti-atrophic effects of leucine supplementation in immobilization and the mechanisms involving HDAC4 in the control of muscle mass. First, the results obtained in RNAseq were validated; we demonstrated that both gene (qPCR) and protein (western blotting) expression of HDAC4 increased in our immobilization model and were attenuated by leucine supplementation. We also show that leucine blocks the nuclear migration of HDAC4 in immobilized animals (Alves et al., 2020; attached). These results incited the investigation of the myostatin pathway since the inhibition of HDAC4s can inhibit myostatin signaling due to the increase in follistatin expression (its endogenous inhibitor). Thus, we demonstrate that leucine supplementation normalizes immobilization-induced myostatin expression while causing strong follistatin expression (Cruz A, Ferian A, Alves PKN, et al., 2020; attached). In addition, seeking to identify additional components of the muscle mass control mechanisms involving HDAC4, we have developed an optimized protocol of resistance exercise by electrostimulation in mice (Alves et al., 2021; paper in submission, attached). These results demonstrate a regulatory role of HDAC4 activity on genes essential for the control of muscle mass. Regarding this new phase of the project, we intend to continue with the objectives proposed in the initial plan (FAPESP 2018/24419-4); for that, we will manipulate HDAC4 levels in vivo and address the relationship of HDAC4 and miR-299a. The interaction of this microRNA with the effects of leucine on HDAC4 was identified from the analysis of a miRseq performed in the context of the aforementioned thematic project, in which we identified that miR-299a was down-regulated during immobilization (~50% of control), which effect is completely blocked by leucine. Furthermore, we identified that this microRNA was predicted to target HDAC4 (in silico data). Thus, the second module of this project aims to seek a better mechanistic understanding of the anti-atrophic actions of leucine. (AU)