"Targeting MICU2 and PDK4 to counteract skeletal muscle atrophy" and "Screening and characterization of mitochondrial calcium uptake inhibitors"

This PhD thesis comprises two projects. Here, we aimed to elucidate the role of mtCa²⁺ signalling in skeletal muscle homeostasis through RNA-based approaches and to explore pharmacological modulation of mtCa²⁺ uptake using selective mtCa²⁺ modulators. Project 1 aims to preserve skeletal muscle mass by targeting two complementary regulators of mitochondrial function: mtCa²⁺ uptake, via silencing of the MCU gatekeeper MICU2, and metabolic flux into the TCA cycle, via silencing of PDK4. Using shRNA-mediated gene silencing in skeletal muscle, we demonstrate that MICU2 knockdown increases mtCa2+ levels and induces significant muscle hypertrophy in the TA. Importantly, MICU2 silencing protects skeletal muscle from denervation- and cancer cachexia–induced atrophy. Similarly, silencing of PDK4 promotes muscle hypertrophy and prevents denervation- and cancer-cachexia-induced atrophy. To translate this study into a therapeutic approach, we evaluated RNA-based delivery systems. For this purpose, we used a novel LNP formulation that can deliver RNA into cells. In parallel, the use of a muscle-specific AAV-MYO vector provided enhanced selectivity for skeletal muscle targeting. Overall, these findings establish that modulation of mtCa²⁺ uptake and oxidative metabolism is sufficient to promote muscle hypertrophy and prevent atrophy, supporting mitochondrial signalling as a powerful therapeutic target for the treatment of skeletal muscle wasting. Project 2 focuses on the pharmacological modulation of mtCa²⁺ uptake by the MCU. Given the broad involvement of mtCa²⁺ signalling in various cellular processes, selective MCU inhibition holds significant therapeutic potential, yet current inhibitors are limited to in vitro or ex vivo use. To define structure and activity relationships and guide rational compound optimization, we screened 47 molecules structurally related to the MCU inhibitor, MCUi11. We evaluated their ability to inhibit mtCa²⁺ uptake without altering cytosolic calcium levels and causing mitochondrial depolarization. We showed that Compound 3, Compound 5, and Compound 27 significantly inhibited mtCa²⁺ uptake. Moreover, the effect of Compound 27 is comparable to that of MCUi11; they are effective at various concentrations. These three compounds are structurally very similar to MCUi11, with different modulations in the same moiety, which provides insight into further modifications. As a result, this systematic structure and activity screening establishes a solid framework for the rational optimization of MCU inhibitors.