Modulation of myofibers integrity and function in health and disease – Atrorescue

Muscle dysfunction is implicated in a plethora of human diseases and in sarcopenia, i.e. muscle atrophy with age that constitutes the leading cause of loss of ambulation and independence. This project aims at identifying important pathways for muscle intracellular organization, under physiological and pathological conditions. The function of the muscle fiber (myofiber) is supported by a precise positioning of its organelles and nuclei. Myofibrils generate contractile force under the control of the “excitation-contraction coupling” (ECC) process, based on the interplay between the sarcoplasmic reticulum (SR), a complex network of tubular endoplasmic reticulum (ER), and Transverse (T)-tubules formed by repeated radial invaginations of the plasma membrane. This interplay takes place at specific interactions sites called triads.

Centronuclear myopathies (CNMs) are genetically inherited neuromuscular disorders whose prominent histopathological feature is the abnormal myonuclei positioning at the center of myofibers. The main genes involved in CNMs are myotubularin (MTM1), dynamin-2 (DNM2), amphiphysin-2 (BIN1), and ryanodine receptor (RYR1). Of interest, T-tubule organization and function and autophagic process are impacted in several CNMs, impairing muscle homeostasis. However, the connections between myonuclei centralization, altered autophagy and inefficient ECC remain poorly understood.

We identified SH3KBP1 (SH3 domain-containing kinase-binding protein 1) as a new factor controlling both myonuclear positioning and T-tubule organization. SH3KBP1 scaffolds perinuclear ER through calnexin binding, and contributes to the formation and maintenance of T-tubules. We also evidenced that this protein binds to DNM2. Thus, these two SH3KBP1 partners could contribute to the correct positioning of myonuclei, organization of triads and to proper function of ECC.

In the ATRORESCUE proposal, we aim at characterizing the pathways regulating the organization and functionality of myofibers, focusing on ER and T-tubule remodeling and on associated functions such as autophagy and ECC in physiological or pathological (CNM) contexts. We aim to decipher how the recently identified SH3KBP1 controls these processes in conjunction with MTM1, DNM2 and BIN1.

To do so, we will use in vitro cellular assays (cultured myotubes and mature myofibers) and in vivo mouse models (main canonical forms of CNMs linked to mutations in Mtm1, Bin1 or Dnm2) that were validated by the different partners and determine if modulation of these pathways can rescue CNM phenotypes. This proposal is articulated around three complementary axes:
1- ER remodeling and autophagy. We will determine the biological functions of SH3KBP1 and associated proteins in the formation/maintenance of ER, and thus nuclear positioning, through regulation of the autophagic pathway.
2- Triad formation and ECC functionality. We will analyze the impact of SH3KBP1 modulation on the efficiency of T-tubule/triad formation and its repercussions on the ECC.
3- Modulation of SH3KBP1-controlled pathways in mouse models of CNMs. As SH3KBP1 downregulation mimics CNM phenotypes, we will increase its level in three different CNM mouse models through viral transduction and quantify beneficial impacts on muscle functionality with a special focus on myofiber physiology, myonuclei positioning, ER and T-tubule architecture, and ECC efficiency.

This collaborative project involves three research groups gathering complementary skills that will allow the study of SH3KBP1-governed pathways involved in the control of the ER, SR, T-tubules and ECC, in vitro and in vivo. Thus, ATRORESCUE will improve our understanding of skeletal muscle function and regulation. Moreover, the validation of SH3KBP1 modulation as a strategy to rescue CNM related phenotypes in vivo may pave the way to treat other neuromuscular diseases and physiological states associated with altered myonuclei location and triad integrity.