IMPACT OF CLOZAPINE ON ADIPOSE AND SKELETAL MUSCLE CELLS: IMPLICATIONS FOR METABOLIC SIDE EFFECTS

Clozapine (CLZ), a second-generation antipsychotic (SGA), is particularly effective in the treatment of schizophrenia, but its use has been restricted to patients who do not respond to other medications due to severe side effects such as cardiomyopathy, seizures and agranulocytosis, weight gain, insulin resistance, cardiovascular disease and metabolic syndrome through mechanisms that are not yet fully understood. The first chapter of this thesis presents a paper accepted with revision, demonstrating that CLZ inhibits mitochondrial biogenesis by reducing PGC1α expression, mitochondrial DNA content, and mitochondrial mass as early as day 3 (T3). Also, the expression of the mitochondrial main proteins was altered by CLZ treatment. At T3, cells displayed elevated ATP levels, which were partially reduced by rotenone, a complex I inhibitor, suggesting a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Despite the inhibition of mitochondrial biogenesis, the treatment with CLZ does not induce a compensatory increase in glycolytic activity. Instead, CLZ-treated cells show a slight decrease in glycolysis but exhibit enhanced mitochondrial function, characterized by increased expression of electron transport chain complexes, elevated mitochondrial membrane potential, and improved mitochondrial activity. Notably, CLZ stimulates the respiratory capacity linked to ATP synthesis and increases the functionality of complex II. Therefore, CLZ suppresses mitochondrial biogenesis in differentiating SW872 cells, which nevertheless maintained adequate ATP levels using an unexpected strategy which was associated with enhanced oxygen consumption driven by lipid substrate-dependent stimulation of complexes I and II, an event associated with increased mitochondrial membrane potential, ATP synthesis and mitochondrial ROS generation. Building on the findings of Chapter 1, Chapter 2 extends the investigation to myotubes, a cell model with specific metabolic susceptibilities, to further elucidate CLZ-induced metabolic side effects across different cell types that are both implicated in metabolic dysregulation. This study has been the subject of a paper that is now under consideration for publication. It has been reported that exposure to CLZ has also been associated with potentially important adverse reactions in the skeletal muscle cells (Barbosa et al., 2022). Indeed, some reports indicate that CLZ promotes myopathy associated with muscle weakness. Most importantly, the use of CLZ has also been associated with the development of rhabdomyolysis, i.e., a condition characterized by the breakdown of skeletal muscle tissue and the release of myoglobin, into the bloodstream. Although CLZ-induced rhabdomyolysis is a rare event, some clinicians suggest that patients under CLZ treatment should be monitored for the appearance of signs of myopathy (Béchard et al., 2022), especially when concomitantly taking other drugs increasing the risk of rhabdomyolysis (e.g., statins), or with previous history of muscle-related adverse effects. To date, a small number of studies have been carried out on the myotoxic effects of the drug in vitro and on the mechanisms underlying the muscle-related adverse effects. In the study reported in chapter 2, C2C12 derived myotubes were used to fill some of these gaps. The results show that CLZ interferes with myogenic differentiation by being selectively toxic to differentiated myotubes, but not to proliferating myoblasts. According to this evidence, micromolar concentrations of CLZ trigger massive apoptosis in myotubes, an event occurring under conditions in which the drug fails to promote toxic effects in myoblasts as well as in reserve cells. Consistent results were also obtained in primary myotubes. In conclusion, the results reported in this thesis may help to shed light on the detailed molecular mechanisms explaining some of the adverse metabolic effects induced by CLZ.