Endocrine Abstracts

JOINT2852

Bmal1 is the key transcription factor of the molecular circadian clock, whose cyclic expression influences energy metabolism and regulation of cell growth and maintenance. The current study aims to elucidate the role of Bmal1 muscle metabolism regulation, with a focus on mitochondrial activity and lipid accumulation. The murine C2C12 cells differentiated into myocytes (C2C12 WT), and C2C12 in which the Bmal1 gene was deleted through CRISPR/Cas9 genome editing technology (C2C12 KOBmal1) were used. Mitochondrial oxidative capacity and efficiency were evaluated using Seahorse assays and MitoTracker Red CMXRos staining, respectively, in both cells. Simultaneously, the expression levels of DRP-1 protein, a key marker of mitochondrial fission, as well as the subcellular localization of myogenin, a transcription factor involved in muscle differentiation, and of HSP70, a molecular chaperone regulating stress response, were investigated via immunofluorescence. In addition, Bodipy staining was performed in both cell models to monitor potential ectopic lipid accumulation, while gene and protein expression levels of the mTOR pathway, myogenin, C/EBPα and PGC1α, key regulators of muscle anabolism, lipogenesis and mitochondrial biogenesis, were assessed by RT-qPCR and western blot, respectively. Furthermore, irisin levels, a myokine involved in mitochondrial function and muscle health, were quantified in both cell models through ELISA assay. Compared to C2C12 WT , basal respiration was significantly lower (P=0.03) while maximal respiration and reserve respiratory capacity were significantly higher (P=0.009; P=0.003) in C2C12 KOBmal1cells. Morphologically, C2C12 KOBmal1 cells showed a reduction in mitochondrial branching (-18.22%) and a significant decrease in mitochondrial branch length (P=0.01) compared to C2C12 WT cells. These changes were associated with increased DRP1 protein levels (+17%) and a significant reduction in PGC1α gene (P=0.01) and protein (P=0.03) levels. These mitochondrial alterations in C2C12 KOBmal1 were accompanied by significantly increased lipid accumulation (P=0.03) and C/EBPα protein expression (P=0.03), indicating a shift toward lipid storage. A marked downregulation of mTOR (-42.86%), p70S6K (-61.43%), and 4E-BP1 (-38.82%) genes was observed in C2C12 KOBmal1 cells compared to C2C12 WT , alongside reduced levels of myogenin (-27.70%). Immunofluorescence revealed perinuclear distribution of myogenin protein in C2C12 KOBmal1 , unlike its nuclear expression in C2C12 WT concomitantly with increased HSP70 nuclear expression, potentially linking reduced myogenic signalling to cellular stress. Lastly, C2C12 KOBmal1 showed significantly decreased irisin levels (P=0.03), reinforcing the impairment in metabolic balance. Concluding, Bmal1 deletion in myocytes disrupts mitochondrial morphology and function, promoting lipid accumulation and suppressing mTOR-driven anabolism through nuclear HSP70 accumulation.