UNRAVELING A NEW REGULATOR OF THE MCU COMPLEX, CLPB, AND STUDY OF THE ROLE OF MITOCHONDRIAL CALCIUM IN SKELETAL MUSCLE REGENERATION

Mitochondria play a pivotal role in Ca2+ homeostasis, thanks to the presence of the steep mitochondrial membrane potential (ΔΨ) and the presence in the IMM of a highly specialized Ca2+ channel, the Mitochondrial Calcium Uniporter (MCU) complex, enabling the entering of Ca2+ ions into the matrix in response to elevated cytosolic Ca2+ levels upon cell stimulation. Mitochondrial Ca2+ exerts diverse effects, from enhancing oxidative metabolism to modulating cell death pathways. Under pathological conditions, an excess of mitochondrial Ca2+ can induce the opening of the mitochondrial permeability transition pore (mPTP), leading to the release of pro-apoptotic factors in the cytosol and ultimately resulting in cell death. Additionally, mitochondrial Ca2+ overload serves as a crucial trigger for reactive oxygen species (ROS) generation. Given these implications, the MCU complex undergoes meticulous regulation to prevent the adverse effects of mitochondrial Ca2+ overload. Recent studies highlight the key role in this context of the proteolytic regulation provided by the mitochondrial AAA-proteases. Notably, the human caseinolytic peptidase B protein homolog (CLPB), identified as an IMS disaggregase, has been proposed to be implicated in this regulatory process. In this regard, the first aim of my PhD project focused on unraveling the role of CLPB in the regulation of the protein stability of the components of the MCU complex. I decided to focus my attention on CLPB not only for its suggested role as a regulator of the solubility of the MCU regulators MICU1 and MICU2, but also because of its association with a rare mitochondrial disorder characterized by neutropenia and 3-methylglutaconic aciduria. My data demonstrate that CLPB is a crucial regulator of the protein stability of critical MCU complex components and that alterations in mitochondrial Ca2+ are evident also in the pathological context of CLPB-mutated fibroblasts from patients. Future studies will elucidate the physiopathological relevance of this new regulatory process. The second aim of my PhD research was to characterize the role of MCU in satellite cells, the skeletal muscle stem cells, during skeletal muscle regeneration. These cells play a pivotal role in the remarkable regenerative capacity of skeletal muscle following injury. While typically quiescent in resting conditions, satellite cells undergo dynamic responses upon muscle injury, engaging in proliferation, differentiation and fusing to facilitate muscle repair, or returning to quiescence to replenish the stem cell pool. Different metabolic preferences have been associated with the various states of satellite cell activity, including quiescence, activation, and differentiation. However, the influence of the metabolic status on these distinct stages remains unclear. Considering the well-established role of mitochondrial Ca2+ in regulating oxidative metabolism, we hypothesized that it could be a critical factor in the activation and differentiation of satellite cells. To test this hypothesis, we performed both in vitro and ex-vivo experiments on satellite cells pharmacologically inhibiting MCU. The results clearly demonstrate a significant impairment in their differentiation capacity. Additionally, to investigate the role of mitochondrial Ca2+ during skeletal muscle regeneration in vivo, we generated a mouse model knockout (KO) for MCU specifically in satellite cells (MCUsc-/-). Strikingly, we observed a significant impairment in skeletal muscle regeneration post-injury induced by cardiotoxin (CTX) injection. This study holds the potential to shed light on the molecular basis of pathological conditions characterized by satellite cell dysfunction, such as aging.